Pseudoneoplastic lesions of the testis and paratesticular structures

Pseudotumors or tumor-like proliferations (non-neoplastic masses) and benign mimickers (non-neoplastic cellular proliferations) are rare in the testis and paratesticular structures. Clinically, these lesions (cysts, ectopic tissues, and vascular, inflammatory, or hyperplastic lesions) are of great interest for the reason that, because of the topography, they may be relevant as differential diagnoses. The purpose of this paper is to present an overview of the pseudoneoplasic entities arising in the testis and paratesticular structures; emphasis is placed on how the practicing pathologist may distinguish benign mimickers and pseudotumors from true neoplasia. These lesions can be classified as macroscopic or microscopic mimickers of neoplasia.
Introduction
There are many lesions that can simulate a neoplasm in the testis or paratesticular structures. Their incidence among tumors arising within the scrotal sac varies according to different series from 6 to 30% [16, 32]. These pseudoneoplastic lesions can be divided into those that only macroscopically imitate neoplasia (Table 1) and those that microscopically imitate neoplasia, regardless of whether they form a macroscopic mass (Table 2). The latter group causes more problems to the practicing pathologist in terms of the correct classification of a giving lesion. Hereby, we summarize main tumor-like lesions and benign mimickers that may be seen in the testis and paratesticular coverages, with emphasis on morphologic criteria for the differential diagnosis from true neoplasia.
Table 1Macroscopic mimickers (pseudotumors) of testicular and paratesticular neoplasiaVascular lesions  Intratesticular hemorrhage  Segmental testicular infarction  Organizad testicular hematocele    Cholesterol granuloma of the tunica vaginalisInflammatory lesions  Nonspecific infectious inflammatory lesions  Specific infectious inflammatory lesions  Non-infectious inflammatory lesions    Idiopathic inflammatory lesions     Idiopathic granulomatous orchitis     Testicular malakoplakia     Testicular sarcoidosis  Meconium periorchitis  Sperm granulomaCysts  Testicular cysts    Albuginea cysts    Parenchymal cysts (Epidermoid cysts)    Rete-testis cysts- Cystic displasia of the rete testis  Epididymal cysts and Spermatoceles  Spermatic cord cystsEctopic tissues  Adrenal cortical rests  Spleno-gonadal fusionTesticular appendagesMiscellaneous other lesionsFibrous pseudotumors; (Fibromatous periorchitis-Nodular periorchitis)  Amyloidosis  Polyorchididm  Sclerosing lipogranulomaTable 2Microscopic mimickers of testicular and paratesticular neoplasiaTesticular  Inflammatory-reactive lesions   Lymphocitic orchitis (Testicular pseudolymphoma)   Rosai-Dorfman disease  Sertoli cell hyperplasia    Pick adenoma    Hamartomatous proliferation testicular feminization syndrome  Interstitial cell hyperplasia    Leydig cell hyperplasia    Testicular “tumor” of the adrenogenital syndrome  Hyperplasia of the rete testisEpididymis  Adenomatoid hyperplasiaTunicas albuginea-vaginalis  Mesothelial hyperplasiaSpermatic cord  Vasitis nodosa  Inflammatory pseudotumor (Funiculitis proliferans)Miscellaneous other lesions
Macroscopic mimickers (pseudotumors) of testicular and paratesticular neoplasia
This group of lesions refers to those that macroscopically imitate a neoplasia. Frequently, these lesions affect both compartments and are therefore difficult to establish if one or both are affected. For this reason, we classify the tumor-like lesions according to their etiology.
Vascular lesions
The majority of the vascular disorders of the male gonad are not confused with a tumor because, in addition to causing acute symptoms, they usually affect the entire gonad. However, in the rare situation in which the vascular lesion is segmental, it can simulate neoplasia, as follows:
Intratesticular hemorrhage, which occurs spontaneously [24] or in the context of anticoagulation treatment [15], may simulate a tumor at ultrasound examination.Segmental testicular infarction, a lesion clinically characterized by slight local pain unrelated to any acute episode [20], may be related to isolated or systemic vasculitis [43, 103] with morphology of polyarteritis nodosa [21], giant cell vasculitis [94], or Wegener granulomatosis [45]. Up to year 2000, 81 cases of systemic vasculitis with testicular tumor-like lesion have been recorded [45]. Other cases are seen in the context of a hematological disease (sickle cell anemia) [53] or associated with nonspecific perivascular fibrosis. Any of the phases of an infarction can be observed from acute (with hemorrhage) to healing stage (Fig. 1). Currently, the clinical diagnosis can be suspected with Doppler sonography [81], avoiding orchiectomy.Organized testicular hematocele and other hemorrhages in the tunica vaginalis are rarely confused with a neoplasia [89], but this can happen occasionally in long-standing cases because of fibrous thickening with cholesterol granuloma formation in the tunica vaginalis (Fig. 2) [61] Exceptionally, a true testicular neoplasia presents clinically with a hematocele [86].Fig. 1Segmental testicular infarction. a Acute hemorrhagic with reinforcement of the peripheral area (arrows). b Healing fibrosisFig. 2Cholesterol granuloma in the tunica vaginalis (H&E)
Inflammatory lesions
Similar to what was stated for vascular tumor-like lesions, inflammation that simulates a neoplasia usually has atypical clinical features; these entities can be grouped as follows:
Nonspecific infectious inflammatory lesions with a tumor-like presentation are frequently chronic processes causing progressive fibrosis, which may clinically [38] or sonographicaly [23] simulate neoplasia. Rarely a testicular, epididymal, or vas deferens abscess can look like a neoplasia [84]. Very occasionally, testicular neoplasia can clinically imitate an acute inflammatory process [44].Specific infectious inflammatory lesions. The entities of this group that most often have been confused with neoplasias are granulomatous inflammation in tuberculosis [87], brucellosis [49], syphillis [5], fungal infections [42], and parasite diseases [9]. These lesions are usually not problematic for the pathologist.Noninfectious inflammatory lesions. This group of tumor-like lesions include different entities among which can be highlighted:Idiopathic granulomatous orchitis, probably of autoimmune aetiology, of which around 230 cases have been published [4], is characterized by tubular granulomas (tubular orchitis; Fig. 3) or interstitial granulomas (interstitial orchitis). The presence of intratubular giant cells differentiate this entity from infectious granulomatous orchitis [70]. Diffuse testicular hypoechoic involvement with only peripheral low-resistance flow on color Doppler sonography is a typical but not pathognomonic pattern [72].Malakoplakia. This lesion is secondary to a mononuclear decrease in cyclin–guanine monophosphate that impairs the killing of bacteria. Fusion of the phagolysosomes with bacterial rests produces the characteristic Michaelis–Gutman bodies in the cytoplasm of the macrophages (von Hansemann cells) [1] (Fig. 4). Giant cells are occasional or absent. Testicular involvement represents only 12% of genital malakoplakia with around 388 cases in the literature [19, 56]; exceptionally, it may affect the epididymis only [31]. It has been related to idiopathic granulomatous orchitis [56, 57] and chronic xantogranulomatous inflammation of testis [85, 109, 110]; this last also reported in the spermatic cord [64, 100].Sarcoidosis. Testicular involvement in a systemic sarcoidosis [39] is exceedingly rare, and its presentation as the primary form is even rarer; in these cases, the epididymis is affected more with the testicle being involved by contiguity [83].Meconium periorchitis. This is an infrequent lesion (around 30 cases being reported) that typically presents in the first months of life; most times there is a clinical history with obstetric problem that has caused the passing of meconium toward the testicular surrounding structures [29]. The macroscopic appearance is a myxoid material with calcified pearls resulting from the calcification of the remains of squamous cells or lanugo hairs [107] (Fig. 5). Suspicion of neoplasia, although very uncommon in this period of life, may be caused by a scrotal mass or sonographically detectable calcifications [6]. Clinically, the peritesticular and spermatic cord enlargement can simulate a paratesticular rhabdomyosarcoma.Sperm granuloma. As its name indicates, this granulomatous lesion with few giant cells is the consequence of extravasation of spermatozoa generally postvasectomy (40% of vasectomized men and 2.5% of general population) [108]. When it produces a tumor-like lesion, it is usually located in the deferent duct or the epididymis [22] with firm nodules of 0.7 to 4 cm with occasional cysts formation.Fig. 3Idiopathic granulomatous orchitis characterized by tubular granulomas (tubular orchitis)Fig. 4Malakoplakia. a Macroscopic appearance with a homogeneous aspect. b The characteristic Michaelis–Gutman bodies in the cytoplasm of the macrophages (von Hansemann cells) (PAS)Fig. 5Meconium periorchitis. a calcified pearls resulting from the calcification of the remains of squamous cells or lanugo hairs. b Keratin rest in one of the calcifications (H&E)
Cysts
The majority of the cysts with a tumor-like appearance are paratesticular structures, but the testicle may occasionally have some cystic lesions that can be confused with a neoplasia.
Testicular cysts occur in approximately 8–10% of patients with a lump in the testis, including those of the tunica albuginea or the parenchyma [33].Tunica albuginea cysts do not usually cause any diagnostic problem, unless if they are complex cysts [74].Parenchyma testicular cysts can be more difficult to distinguish from a neoplasia and if there is the slightest suspicion of an intracystic content, one must suspect a malignant neoplasia [107]. Special consideration deserves the testicular Epidermoid cyst, which must only be lined with squamous epithelium (Fig. 6). It is recommended that the specimen is examined “in toto” to avoid underdiagnosis of any area of teratoma (especially among postpuberal patients) or intratubular germ cell neoplasia, as sonographically it is not possible to distinguish between these lesions [54, 106]. Epidermoid cysts represent 1% of the masses of the testes. Recent genetic studies have shown that there is no chromosome 12p abnormality [14], thus supporting its distinction from teratoma.Tubular ectasia of the rete testis secondary to obstruction and generally located in the mediastinum area of the testes (Fig. 7) [69], is usually bilateral and very different from cystic dysplasia of the rete testis, a congenital lesion with complete testicular parenchyma substitution [68].Epididymal cysts and spermatoceles are relatively frequent and the majority are in relation to the inflammatory processes. The differential diagnosis with other entities are related to its size, and similar to the other cystic formations a true neoplasia must be considered in case of observing any content in its interior [108].Spermatic cord cysts. The majority of these cysts do not cause any diagnostic doubts, only the occasional Epidermoid cysts [46, 105] can simulate a neoplasia. These cysts can be unilocular or multilocular depending on their origin [65], and the multilocular must be distinguished from the exceptional cystadenomas of probably Müllerian origin [55].Fig. 6Epidermoid cyst, which must only be lined with squamous cell epithelium (H&E)Fig. 7a Tubular ectasia of the rete testis located in the mediastinum area of the testes (arrows). b Microscopic aspect (H&E)
Ectopic tissues
Ectopic adrenocortical tissue is relatively frequent in the tunica albuginea, rete testis, epididymis, and spermatic cord and occasionally reaches the size to be symptomatic. Its incidence ranges from 2.5 to 15% [17, 102]. It is made up of adrenal cortical nodes surrounded by a connective tissue band and of about 5 mm in diameter on average (Fig. 8); for which reason, they are not clinically palpable. Only in cases of congenital adrenal hyperplasia or Cushing syndrome, ectopic adrenocortical tissue can be prominent and appear as a tumor-like lesion [90].
Splenic–gonadal fusion, as its name indicates, is the fusion of spleen and gonad. It is more frequent on the left side, with about 148 published cases [41]. Morphologically, the ectopic splenic tissue can be in close relation to the head of the epididymis or the upper pole of the testis (Fig. 9) or being separated from it; likewise, there may or may not be a structural continuity between the normal spleen and the ectopic tissue [30]. The same alteration has been described in women, but it is much less frequent that in men, probably because it is much easier to find it clinically in men. In about 30% of the cases, it is associated with complex malformations such as micrognathia, peromelia, or phocomelia (absence of upper portion of a limb) [30, 60, 95]. In three of the reported cases, the fusion was associated with a germ cell tumor of the testis [41].
An exceptional hepato-gonadal fusion is reported [26].
Fig. 8
Ectopic adrenocortical tissue. Adrenal cortical nodes surrounded by a connective tissue band in continuity with epididymis. (H&E)
Fig. 9
Splenic–gonadal fusion. The ectopic splenic tissue in close relation with the upper pole of the testis (H&E)
Testicular appendages
There are five testicular appendages, but for the surgical pathologist, only three can be of interest (appendix testis or hydatid of Morgagni, appendix epididymis, paradidymis or organ of Giraldes; Fig. 10). These structures are not usually the origin of a tumor-like lesion, but in rare cases of large-sized cysts, it may presents as a paratesticular mass [93]. A case of ectopic epididymal tissue in an appendix testis [101] has recently been reported.
Fig. 10Normal testicular appendages
Miscellaneous other lesions
Fibrous pseudotumors. This name refers to a fibrosis phenomenon with paucicellular hyalinized collagen (Fig. 11) presenting as nodular (single or multiple) or diffuse lesion of the testicular tunics [96, 71]. Sometimes, a node can be free (scrotal mouse) [108]. This broadly considered spectrum of lesions has received diverse names: chronic periorchitis, fibromatous periorchitis, nodular periorchitis, fibrous proliferation of the tunica, nonspecific paratesticular fibrosis, granulomatous periorchitis, nodular fibrous pseudotumor, fibrous pseudotumor, inflammatory pseudotumor, fibroma, reactive periorchitis [108], indicating its controversial pathogenesis. Some cases have been reported preceded by trauma or infection, and on occasion, an inflammatory component can be observed and granulation tissue suggesting the possibility that there might be the healing of an inflammatory pseudotumor, (which will be described later on) [10]. Although radiologically, it is not difficult to recognize, upon occasion, an intraoperative frozen section becomes necessary.
Amyloidosis. It is usually bilateral and present in a patient with a prior history of amyloidosis [34]; more rarely, it is a primary form that by being a cryptorquidic patient simulates a testicular tumor [13].
Polyorchidism or supernumerary testes is a rare condition, which is easy to recognize. However, the sonography can occasionally be different from that of the normal testis, and the condition may then be tumor suspicious [79].
Fig. 11
Fibrous pseudotumor. a Well outlined fibrous-like nodule. b Paucicellular hyalinized collagen with calcification
Microscopic mimickers of testicular and paratesticular neoplasia
Lesions or cellular changes that microscopically imitate a neoplasia are included under this category, whether or not they make a clinical mass. These changes are closely related to the structure of the organ in which they arises, as follows.
Testicular
Inflammatory-reactive lesions Some lesion, already described above such as xantogranulomatous orchitis, idiopathic granulomatous orchitis and malakoplakia could be considered under this category; their microscopic confusion with a neoplasia (seminoma for example) is not currently a usual event; therefore, we preferred to include them in the macroscopic mimickers (pseudotumors). The situation is different with lymphocitic orchitis or testicular pseudolymphoma [2, 3], which is characterized by a lymphocytic and plasmocellular reaction that may be confused with a lymphoma, but immunohistochemistry will show that the cellular infiltrate is polyclonal (Fig. 12). Among these idiopathic lesions, we can include Rosai–Dorfman disease; histological examination of the testicular mass reveals an inflammatory lesion comprising lymphocytes, plasma cells, and sheets of pale staining histiocytes, some containing lymphocytes within their ample cytoplasm, suggestive of emperipolesis. The histiocytes stained positive for CD68, S100 by immunohistochemistry and negative for CD1a, while ultrastructural examination confirmed emperipolesis [25].
Fig. 12Lymphocitic orchitis. Lymphocitic cellular infiltration of polyclonal type. a CD45RA (lymphocytes B). b UCHL-1 (lymphocytes T)
Sertoli cell hyperplasia In a series of situations, nonencapsulated nodules of Sertoli cells can be found, especially known in cryptorchid testes as Pick’s adenomas (Fig. 13a) [77]. Because of their appearance, these should be distinguished from the actual Sertoli cell tumors that generally are larger and sometimes there are areas that mimic Call–Exner bodies (Fig. 13b) A differential diagnosis with a yolk-sac tumor is not usually in the scope, but the immunohistochemistry study with AFP, calretinin, α-inhibin, and CD 99 can help [40].
Fig. 13Sertoli cell hyperplasia. a Nonencapsulated nodules of Sertoli cells (Pick’s adenoma). b Sometimes there are areas that mimic Call–Exner bodiesA special consideration merit the androgen insensitivity syndrome or testicular feminization (male pseudohermaphrodism, caused by a failure of androgen receptor binding) that in 63% of cases can have tubular hamartomas (tubules lined by immature Sertoli cells) [82] that must be differentiated from the Sertoli cell adenomas and sex cord tumor with annular tubules [76].
Interstitial cell hyperplasia In testicles with marked tubular atrophy, such as in the Klinefelter’s syndrome, it is possible to see Leydig cell nodules that must be distinguished from Leydig cell tumors. An interstitial growth without expansive pattern favors hyperplasia (Fig. 14) [62].
Fig. 14Leydig cell hyperplasiaNodules of eosinophilic cells appearing to be Leydig cells are found in the patients with adrenogenital syndrome [18, 48] and Nelson’s syndrome (adrenocorticotropic hormone-secreting pituitary adenoma after bilateral adrenalectomy), some of this last syndrome with excessive testosterone production [91]; proof that these interstitials cells are not only morphologically similar to Leydig cells but also have the functional property of these cells. These nodules are usually bilateral and of a large size with cellular pleomorphism and pigmentation. The clinical history and a complete endocrinological profile avoid an unnecessary orchiectomy [78] because only one case of aggressive behavior is published [18].
Hyperplasia of the rete testis The normal rete testis epithelium is flat, but in some hyperestrogenic situations (treatment or hepatic dysfunction), the epithelium may become columnar and rarely a micropapillary growth of bland cells can be observed. The diagnosis of rete testis hyperplasia is subjective, and adenomatous lesions are rarely seen [35, 63]. In some cases, there are intracytoplasmic hyaline eosinophilic globules resembling a yolk sac tumor, but the negative stains of α-fetoprotein or placental alkaline phosphatase help to rule this differential diagnosis [99].Pseudohyperplasia of the rete testis and epithelial reaction in case of germ cell invasion and cryptorchidism must be differentiated from real hyperplasia of the rete testis [62].
Epididymis
Benign microscopic mimickers of cancer in the epididymis are very rare. Cysts may occur but do not resemble tumors microscopically. However, some cases of adenomatoid hyperplasia of the rete testis can involve the epididymis, and occasionally, they may become macroscopically apparent [93].
Tunica albuginea and vaginalis
Non-neoplastic mesothelial lesions involving the paratesticular region include mesothelial cysts and reactive mesothelial hyperplasia [73].
Mesothelial hyperplasia is the most important benign mimicker of the testicular tunics. It is present as a reactive lesion in hydrocele or hernia but may also be found microscopically in older men [80]. The mesothelial proliferation has an epithelial appearance, and rarely, a spindle cell proliferation can be present. In the differential diagnosis with mesothelioma, the bland nucleus, no true invasion, and associated inflammatory elements can be useful (Fig. 15) [11, 98]. Recently, a case was published with “atypical” mesothelial hyperplasia on one side and “well differentiated” mesothelioma on the contalateral [97]. This case is an example of the subjective interpretation of some mesothelial proliferative lesions because some malignant mesotheliomas lack cellular atypia. In these cases, an extensive confluence or prominent infiltration favors a malignant diagnosis. Unfortunately, the immunohistochemical expression of benign and malignant mesothelial proliferations are similar (low- and high-molecular-weight cytokeratins and vimentin) [11, 28], and only the metastatic neoplasias can be differentiated by the CEA, Ber-EP4, and B72.3 expression [27]. Only deoxyribonucleic acid ploidy can distinguish some borderline lesions [28].
Fig. 15Mesothelial hyperplasia. The bland nucleus, no true invasion and associated inflammatory elements, can be useful to distinguish from malignant mesothelioma
Spermatic cord
The vas deferens and the soft tissues of spermatic cord can have benign mimickers.
Vasitis nodosa is a ductular proliferation, generally after vasectomy [36], although it can be seen following other trauma on that area, [75]. It has a microglandular morphology (Fig. 16) with mild nuclear atipia and perineural growth [7] or benign vascular invasion [8] that may be mistaken for malignancy [104]. The frequent hyperplasia of nerve fibers in the adventitia can explain the painful symptoms in some patients [36]. The coincidence with microscopic sperm granulomas and inflammatory reaction can help in the correct diagnosis. An analogous epididymal lesion also occur [88].Proliferative funiculitis is the inflammation of the spermatic cord usually the result of an extension of vasitis. The soft tissues of the spermatic cord are also the most common site of an inflammatory pseudotumor in the male genital tract [37, 50, 58]. The lesion is ill defined, myxoid with white-gray color, and a moderate cellular proliferation with loose collagen fibers and irregular infiltration of inflammatory cells. An exceptional case has been reported that was largely infiltrated by mast cells [92]. In some cases, a prominent spindle cell proliferation mimics a sarcoma (pseudosarcomatous myofibroblastic proliferation), but low mitotic index, a capillary pattern, inflammatory cells, and absence of atypical mitoses speak against a diagnosis of sarcoma. Unfortunately, immunohistochemistry is only partly helpful because the cells express actin and vimentin, less strongly desmin and exceptionally cytokeratin. Two cases of this lesion in epididymis [12, 51] and one in the rete testis [47] have been reported.Fig. 16Vasitis nodosa. Ductular proliferation with a microglandular morphology
Embryonic remnants
Although ectopic tissue usually is more problematic for constituting a mass, some of them can cause microscopic diagnostic doubts for which reason they can be included in this group of benign mimics. The presence of seminiferous tubules within the tunica albuginea [67], Leydig cells in rete testis, albuginea, spermatic cord, or within sclerotic tubules [59, 66], prostate gland in the epididymis [52], and special circumstances with muscular rete testis hypertrophy [27] can mimic a neoplasia.
The lesions described constitute a large heterogeneous group, without etiological or pathogenic relations among them or with true neoplasias. However, in spite of that, the patient with one or more of these tumor-like and/or benign mimickers can have a concomitant or ulterior true neoplasm.

testis
paratesticular structures
pseudotumor
tumor-like
benign mimics


The effect of acute hyperglycemia on retinal thickness and ocular refraction in healthy subjects

Purpose To quantify the retinal thickness and the refractive error of the healthy human eye during hyperglycemia by means of optical coherence tomography (OCT) and Hartmann–Shack aberrometry.
Patients with diabetes mellitus (DM) often experience subjective symptoms of blurred vision associated to hyperglycemia. The nature and origin of this phenomenon are still unclear. Blurred vision during hyperglycemia could be a result of transient refractive alterations due to changes in the lens [5, 12, 15, 25, 27, 36, 37], but it could also be caused by changes in the retina. Macular edema, or retinal thickening due to abnormal fluid accumulation within the macula, is a common cause of visual loss [1, 14, 22]. The degree of retinal thickening has been found to be significantly correlated with visual acuity [24]. Furthermore, a change in retinal thickness, resulting in a change in axial eye length, could also induce a change in ocular refractive error. For instance, it can be calculated that with a 50 μm increase in retinal thickness, the ocular refractive error becomes 0.15 D more hyperopic [30].
Several studies have demonstrated that retinal thickness is affected by DM [2, 7, 8, 18, 21, 23, 26, 32, 33, 35, 38]. In general, an increase in retinal thickness has been reported in patients with long-term DM and advanced stages of retinopathy [7, 8, 18, 32, 33, 35]. However, in diabetic patients with and without minimal diabetic retinopathy, a decrease in retinal thickness has been observed [2, 23]. In healthy subjects, it has been shown that the average retinal thickness did not change during normo-insulinaemic hyperglycemia [13].
It is unclear whether the thickness of the different retinal areas, such as the foveal area, the pericentral foveal area, and the peripheral foveal area, changes during acute hyperglycemia and suppression of insulin. A change in retinal thickness and/or ocular refractive error could explain the subjective symptoms of blurred vision in patients with DM and hyperglycemia. Therefore, in the present study the effect of hyperglycemia on retinal thickness and ocular refractive error was investigated in healthy subjects during suppression of endogenous insulin. Retinal thickness was measured by means of optical coherence tomography (OCT), which is a non-invasive technique that provides cross-sectional retinal images, and produces an objective measurement of the retinal thickness, independent of the refractive status of the eye [10, 11, 29]. Furthermore, aberrometry was used to measure the ocular refractive error. This technique makes it possible to detect small changes in ocular refraction [19].
Subjects and methods
Five healthy subjects (two males and three females) participated in the study. The mean age of the subjects was 24.8 years (range 21.2–32.6), and their mean body mass index (BMI) was 24.2 kg/m2 (range 21.4–29.7). The subjects were screened during a first visit, which included medical history-taking, a physical examination (measurement of visual acuity, weight, height and blood pressure) and collecting a fasting blood sample. Exclusion criteria were a history of DM (or a fasting plasma glucose >5.5 mmol/l), a BMI of >30 kg/m2, elevated blood pressure (>140 / 85 mmHg), a visual acuity of <0.5 (Snellen) or a history of ocular pathology. The investigators of the ocular parameters (NW and MD) were not informed about the blood glucose levels. Furthermore, the investigators who induced hyperglycemia (EE and SS) were not informed about the results of the ocular measurements. The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center in Amsterdam, and written informed consent was obtained from all subjects after the purpose and nature of the study had been explained to them.
Procedure to induce hyperglycemia
After a 10-hour overnight fast, the subjects were given a subcutaneous injection of a low dose (100 μg) of synthetic somatostatin (Sandostatin, Novartis, Basel, Switzerland) in order to suppress endogenous insulin secretion. Each subject underwent an oral glucose tolerance test (OGTT) (75 g glucose) 30 minutes after the somatostatin injection, and blood glucose levels were measured with a blood glucose analyzer (HemoCue Diagnostics BV, Oisterwijk, the Netherlands). Endogenous insulin levels were measured by means of immunometric assays (Luminescence, Bayer Diagnostics, Mijdrecht, the Netherlands) in the Endocrinology Laboratory at the Department of Clinical Chemistry of the VU University Medical Center. The subjects remained in fasting state during the entire procedure.
Ocular measurements
Retinal thickness was measured with the Stratus OCT (Model 3000, Carl Zeiss Meditec, Dublin, CA, USA), which combines a low-coherence scanning interferometer (wavelength 820 nm) with a video camera to visualize the fundus of the eye. The fast macular thickness OCT scan protocol was used to obtain six cross-sectional macular scans, 6 mm in length, which were positioned at equally spaced angular orientations (30°) centred on the fovea. The cross-sectional images were analyzed with OCT3 mapping software that uses an edge-detection technique to locate the vitreoretinal interface and the anterior surface of the retinal pigment epithelium. Retinal thickness was defined as the distance between these two surfaces. Two OCT scans were made of each subject before, and every 30 minutes during the period of hyperglycemia. In order to quantify the retinal thickness, the foveal map constructed by the software was divided into nine Early Treatment Diabetic Retinopathy Study (ETDRS) areas [6]: the central fovea (central circle with a diameter of 1 mm), and the pericentral area (donut-shaped ring with an inner diameter of 1 mm and an outer diameter of 3 mm) and peripheral area (donut-shaped ring with an inner diameter of 3 mm and an outer diameter of 6 mm), both of which were divided into four quadrants. Retinal thickness was calculated for all separate areas, and for the average pericentral and peripheral regions.
Ocular refractive error was determined with an IRX3 aberrometer (Imagine Eye Optics, Paris, France), which performs wavefront analysis of the eye according to the Hartmann-Shack principle [19]. After pupil dilation and paralysis of accommodation with 1.0% cyclopentolate and 5.0% phenylephrine eye-drops, a series of three aberrometry measurements was made before, and every 30 minutes during the hyperglycemic condition. From these measurements, the equivalent refractive error was calculated as: equivalent refractive error (ERE) = sphere + (cylinder / 2).
The measurements at baseline and during maximal hyperglycemia were analyzed, and any change was considered to be meaningful if the difference between the measurements was greater than the threshold of 50 μm for retinal thickness and 0.2 diopters (D) for ERE. The threshold of 50 μm exceeded the 95% confidence interval for the detection of a change in retinal thickness, which has been reported to be approximately 40 μm [4, 20, 28]. A refractive change of more than 0.2 D also surpasses the precision (defined as 95% confidence interval) of the aberrometer for measuring sphere, cylinder, and consequently ERE [3, 31]. In each subject, the significance of a change was obtained from the precision of the measurement instruments and the difference in the ocular parameters at baseline and during hyperglycemia. In the whole group, the significance of a change could be determined by means of Wilcoxon matched pairs signed rank sum tests. P-values ≤0.05 were considered to be statistically significant.
Results
The changes in blood glucose after the administration of somatostatin and glucose are shown in Fig. 1. Mean blood glucose levels rose from 4.0 mmol/l (range 3.4–4.5 mmol/l) to 18.4 mmol/l (range 16.1–22.0 mmol/l) after the OGTT. Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1 pmol/l (range 0.4–4.5 pmol/l), and remained below basal secretion level (<110.0 pmol/l) for 147 minutes (range 75–270 minutes). Subject 01 had a delayed elevation of blood glucose level, compared to the other subjects. This person received a second 75 g oral glucose load after 30 minutes in order to induce a rise in the blood glucose level. In all subjects, the blood glucose and endogenous insulin levels normalized within 6 hours after the OGTT.
Fig. 1Graph of normalized blood glucose levels in the five subjects after the administration of somatostatin and glucose. Data are normalized by subtracting the value at baseline from the measured value in each subject. The oral glucose load (75 g) was administered at T 0. Subject 01 received an extra 75 g oral glucose load at T 30
Figure 2 shows the normalized ERE of the five subjects during hyperglycemia. Mean ERE at baseline was 0.6 D (SD 0.6) and 0.7 D (SD 0.6) during maximal hyperglycemia; no significant change was found in the group as a whole. A small, but significant hyperopic shift of 0.4 D (SD 0.2) in ERE was measured in subject 01 during maximal hyperglycemia, compared to the start of the procedure (p < 0.001). No significant change in ERE was found in any of the other subjects. Normalized retinal thickness parameters are shown in Fig. 3a (central foveal area), Fig. 3b (average pericentral foveal area) and Fig. 3c (average peripheral foveal area). Average central foveal thickness, average pericentral foveal thickness, and average peripheral foveal thickness at baseline were 202 μm (SD 8), 277 μm (SD 5), and 243 μm (SD 8). During maximal hyperglycemia, average central foveal thickness, average pericentral foveal thickness, and average peripheral foveal thickness were 203 μm (SD 7), 275 μm (SD 3), and 242 μm (SD 9). No significant differences were found in the group as a whole. Furthermore, none of the subjects had any significant changes in the thickness of the central fovea, the pericentral fovea, or the peripheral fovea during maximal hyperglycemia, compared to baseline. The nine separate ETDRS areas were not affected by hyperglycemia. At baseline and during hyperglycemia, any change in retinal thickness that occurred in the various areas was less than 15 μm, which was within the previously defined threshold of 50 μm.
Fig. 2Graph of the normalized equivalent refractive error (ERE) in diopters (D) of the five subjects. Data are presented as mean±SD; three measurements were made of each subject every 30 minutes during the procedure. Data are normalized by subtracting the value at baseline from the measured value in each subject. The oral glucose load was administered at T 0. * Significant difference between ERE at T 0 and T 210 (maximal hyperglycemia), p < 0.001Fig. 3Graphs and maps of normalized retinal thickness parameters in the five subjects during hyperglycemia: (a) central fovea, (b) pericentral fovea, (c) peripheral fovea. Data are normalized by subtracting the value at baseline from the measured value in each subject. Each measured area has been indicated by a dark grey area on the retinal maps. No significant changes in retinal parameters were found in any of the subjects. The oral glucose load was administered at T 0
Discussion
Blurred vision is a symptom that occurs frequently in patients with DM and hyperglycemia. The underlying mechanism is still unclear, and therefore the present study was carried out in an attempt to identify a possible cause of this symptom. The effect of reproducible hyperglycemia on retinal thickness and refractive error was studied in healthy young subjects who did not suffer from the systemic effects of DM.
No changes in the thickness of the central, pericentral or peripheral foveal areas were found in any of the subjects during hyperglycemia. In addition, no significant change was measured in any of the nine different ETDRS areas of the macula. In their study, Jeppesen et al. [13] also found no significant difference in retinal thickness in healthy subjects during normo-insulinaemic hyperglycemia. Before and 180 minutes after the start of a hyperglycemic clamp they measured the average thickness of the retina, and found that retinal thickness was not affected by hyperglycemia. Although in the present study retinal thickness was measured under different circumstances than in the study of Jeppesen et al. (hypo-insulinaemic hyperglycemia instead of normo-insulinaemic hyperglycemia), the results confirm those of Jeppesen et al.
Retinal thickness has been reported to change in patients with long-term DM and retinopathy. A morphological change in the retina may even occur in the early stages of diabetic retinopathy [2, 7, 8, 18, 21, 23, 26, 32, 33, 35, 38]. These changes in retinal thickness are usually due to abnormal fluid accumulation resulting from a breakdown of the blood-retinal barrier [34]. Goebel et al. [8] measured retinal thickness by means of OCT in 136 patients with different stages of diabetic retinopathy and with a mean DM duration of 16 years. Mean foveal thickness was 307 ± 136 μm in the diabetic subjects, compared to 153 ± 15 μm in healthy subjects. It seems that only long-term hyperglycemia and/or long-term fluctuations in blood glucose levels have any significant influence on the blood–retina barrier and retinal thickness. From the findings of the present study, it appears that the blood–retina barrier does not seem to be affected by a single episode of acute hyperglycemia. Nevertheless, the fact that no change in retinal thickness could be determined does not exclude the possibility that there could be early dysfunction of the blood retina barrier. Other means of examination could evidence such a dysfunction of the blood retina barrier following acute hyperglycemia.
A factor that could have biased the results of this study is the administration of a synthetic somatostatin analogue to the subjects. Somatostatin is a peptide hormone that inhibits several hormones, including IGF-1 and insulin. IGF-1 is a growth factor that is produced by the hypoxic retina to mediate angiogenesis, resulting in neovascularisation. Somatostatin analogues not only inhibit neovascularisation in patients with advanced diabetic retinopathy, but also stabilize the blood–retinal barrier in patients with diabetic macular edema [16, 17]. It could have been possible that in the present study an increase in retinal thickness during hyperglycemia was prevented by somatostatin. Nevertheless, the efficacy of synthetic somatostatin in the treatment of advanced diabetic retinopathy was investigated by Grant et al. [9]. With maximally tolerated doses of somatostatin (ranging from 200 to 5000 μg/day), after a period of 15 months one out of 22 eyes required panretinal photocoagulation, compared to nine of 24 eyes that were not treated with somatostatin. From the results of the Grant et al. study, it seems that only frequent, large doses of somatostatin over a long period of time have any significant effect on the progression of diabetic retinopathy. Although the effect of somatostatin on the healthy retina has not been investigated yet, it seems to be unlikely that the results of the present study were biased by the administration of one single, low dose (100 μg) of somatostatin.
In conclusion, the results of this study indicate that in healthy subjects, hyperglycemia does not cause any change in retinal thickness. Furthermore, ocular refraction in general was not affected by hyperglycemia. However, there were interindividual variations, as illustrated by subject 01, who had a hyperopic shift of refraction during hyperglycemia. Therefore, it seems that a refractive change during hyperglycemia cannot be explained by a change in retinal thickness. It could well be that other refractive components, such as the lens, are involved in causing blurred vision and refractive alterations during hyperglycemia.

hyperglycemia
refractive errors
optical coherence tomography
diabetes mellitus


Rab proteins and Rab-associated proteins: major actors in the mechanism of protein-trafficking disorders

Ras-associated binding (Rab) proteins and Rab-associated proteins are key regulators of vesicle transport, which is essential for the delivery of proteins to specific intracellular locations. More than 60 human Rab proteins have been identified, and their function has been shown to depend on their interaction with different Rab-associated proteins regulating Rab activation, post-translational modification and intracellular localization. The number of known inherited disorders of vesicle trafficking due to Rab cycle defects has increased substantially during the past decade. This review describes the important role played by Rab proteins in a number of rare monogenic diseases as well as common multifactorial human ones. Although the clinical phenotype in these monogenic inherited diseases is highly variable and dependent on the type of tissue in which the defective Rab or its associated protein is expressed, frequent features are hypopigmentation (Griscelli syndrome), eye defects (Choroideremia, Warburg Micro syndrome and Martsolf syndrome), disturbed immune function (Griscelli syndrome and Charcot–Marie–Tooth disease) and neurological dysfunction (X-linked non-specific mental retardation, Charcot–Marie–Tooth disease, Warburg Micro syndrome and Martsolf syndrome). There is also evidence that alterations in Rab function play an important role in the progression of multifactorial human diseases, such as infectious diseases and type 2 diabetes. Rab proteins must not only be bound to GTP, but they need also to be ‘prenylated’—i.e. bound to the cell membranes by isoprenes, which are intermediaries in the synthesis of cholesterol (e.g. geranyl geranyl or farnesyl compounds). This means that isoprenylation can be influenced by drugs such as statins, which inhibit isoprenylation, or biphosphonates, which inhibit that farnesyl pyrophosphate synthase necessary for Rab GTPase activity. Conclusion: Although protein-trafficking disorders are clinically heterogeneous and represented in almost every subspeciality of pediatrics, the identification of common pathogenic mechanisms may provide a better diagnosis and management of patients with still unknown Rab cycle defects and stimulate the development of therapeutic agents.
Introduction
Germline mutations in the genes involved in the Ras–mitogen-activated protein kinase (MAPK) pathway explain a number of neuro–cardio–facio–cutaneous syndromes, as reported in a paper recently published in this journal [10].
Ras-associated binding (Rab) proteins are small GTPases of the Ras superfamily that continuously cycle between the cytosol and different membranes. In 2002, Pfeffer described 20 different Rab GTPases that have been identified as prenylated proteins localized in distinct membrane-bound compartments [25]. Prenylated proteins are proteins bound to isoprenes, which are intermediaries in the cholesterol synthesis. At the time of the writing this article, more than 60 human Rabs had been identified [26]. This high number of different Rab proteins highlights their importance in the regulation of vesicle trafficking processes, including vesicle formation, motility, tethering and fusion to the acceptor membrane and signaling to other organelles. The ability of Rabs to perform these different tasks in a co-ordinated and regulated manner originates in their highly dynamic conformation and mobility and from their interaction with different Rab-associated proteins, as illustrated in the Rab GTPase cycle (Fig. 1). Rabs can cycle between the active GTP-bound and the non-active GDP-bound forms, assisted by different Rab-associated proteins, such as the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins), respectively [24, 26]. This conformational cycle is the main driving force for the on/off ‘switch’ mechanism of Rabs and their ability to both regulate binding to downstream effectors and perform the desired function. Rabs are initially synthesized as soluble proteins in the cytosol where they are first recognized by a soluble chaperone-like protein named REP (Rab escort protein) [3]. The REP brings the Rab to the RabGGT (Rab geranylgeranyl transferase) for the addition of—generally—two geranylgeranyl groups. These prenyl groups are added, by covalent thioether bonds, to cysteine residue(s) located at the C-terminus of the Rab protein [7]. This post-translational modification of Rabs is needed to allow for the attachment of the Rab proteins into the lipid bilayer of the organelle; consequently, Rabs are considered to be peripheral membrane proteins (Fig. 1). After performing their function, Rab proteins are extracted from the membranes by RabGDI (GDP dissociation inhibitor), and they remain in the cytosol until they are needed again [7, 24, 37]. Statins are known to inhibit protein isoprenylation. As such, they are able to reduce the production of amyloid-beta and inhibit preamyloid catabolism in the lysosomes, which suggests their possible application in the treatment of Alzheimer [20]. However, the therapeutic role of statins in Rab-related disorders has not yet been established. The potent nitrogen-containing biphosphonates, such as pamidronate, alendronate, ibandronate and zoledronate, inhibit a key enzyme, farnesyl pyrophosphate synthase, which is necessary for Rab activity, hence their inhibitory action on osteoclast activity [29].
Fig. 1The Ras-associated binding (Rab) protein–GTPase cycles. Rab proteins are intrinsically soluble and require a post-translational modification for membrane association. They first associate with a Rab escort protein (REP) and form a stable complex that is the substrate for the subsequent dual prenylation of C-terminal cysteine motifs via Rab geranylgeranyl transferase (RabGGT). RabGGT consists of two different functional subunits (RabGGTα and RabGGTß). After lipid tranfer, REP delivers the prenylated Rab to the donor membrane (the REP cycle is shown with blue arrows). In the absence of REP or RabGGT, Rab proteins remain in the cytosol in an inactive state. The transfer of Rab proteins between acceptor and donor membranes is facilitated by the GDP dissociation inhibitor (GDI) (the GDI cycle is shown with red arrows). Both REP and GDI bind the GDP-bound inactive form of Rab. After REP or GDI dissociate from Rabs at the donor membrane, Rabs can cycle between the inactive (GDP-bound) and active (GTP-bound) states. Rab proteins are activated by guanine nucleotide exchange factors (RabGEFs) and deactivated by GTPase activating proteins (RabGAPs), which accelerate the slow intrinsic rates of nucleotide exchange and GTP hydrolysis. In the active state, Rabs interact with structurally and functionally diverse effectors, including cargo sorting complexes on donor membranes, motor proteins involved in vesicular transport and tethering complexes that regulate vesicle fusion with acceptor membranes
Evolutionarily conserved Rabs are usually expressed in all cell and tissue types and regulate more fundamental vesicle transport pathways whereas the less conserved Rab family members function in more specific pathways, which are often organ-dependent. Defects in intracellular vesicle trafficking underlie a large variety of human diseases, including pathologies associated with defects in Rabs or Rab-associated proteins [14, 32]. In this review, we deal with the role of different Rab and rab-associated proteins in inherited (Table 1) as well as certain multifactorial human diseases in different pediatric disciplines. In addition, the review will also focus briefly on recent advances in understanding human disease through the study of Rab proteins from in vitro and mouse knockout studies.
Table 1Human monogenic diseases caused by a Ras-associated binding (Rab) protein or Rab-associated protein defectDiseaseOMIMGeneRabRab-associatedDescriptionGriscelli syndrome type I214450MYO5AxAutosomal recessive, albinism, neurological impairmentGriscelli syndrome type II607624RAB27AxAutosomal recessive, albinism, hemophagocytic syndrome, sometimes secondary neurological impairmentGriscelli syndrome type III609227MLPHxAutosomal recessive, albinismChoroideremia303100REP1xX-linked, progressive loss of vision beginning at an early age, and the choroid and retina undergo complete atrophyNon-specific mental retardation300104GDI1xX-linked, affected males show moderate to severe mental retardation, carrier females may also be mildly affectedCharcot-Marie-Tooth disease type IIb608882RAB7xAutosomal dominant, peripheral sensory neuropathy with late onset muscle weakness, foot ulcers and infectionsWarburg Micro syndrome600118RAB3GAP1xAutosomal recessive, microcephaly, microcornea, congenital cataract, mental retardation, optic atrophy, and hypogenitalismMartsolf syndrome212720RAB3GAP2xAutosomal recessive, congenital cataracts, hypogonadism, and mild mental retardation
Rab proteins in skin and hear pigmentation
Griscelli syndrome (GS) is an autosomal recessive disorder that causes partial albinism [15]. There are three variants of this disease: one is a purely hypopigmentation disorder (GS type III), and two, in addition to the pigmentation defect, are also associated with immunological defects (GS type II) or with primary neurological dysfunctions (GS type I). Griscelli syndrome type II with immunological defects is caused by missense mutations in the gene encoding Rab27a [18]. This Rab regulates the movement of melanosomes to the cell periphery of melanocytes and also regulates the secretion of lytic granules in cytotoxic T lymphocytes (CTL) [35]. The lack of Rab27a thus causes pigment anomalies and dysfunctional T lymphocytes, which is in agreement with the defects observed in the patients that present with albinism and hemophagocytic syndrome. The GS type I with neurological symptoms is caused by mutations in the MYO5A gene encoding the motor protein myosin Va [22], a putative Rab27a effector that drives the peripheral distribution of melanosomes along actin filaments [15]. As myosin Va does not participate in the exocytosis of lytic granules of CTL, the inactivation of this protein does not lead to immunological symptoms. Finally, GS type III in patients with typically pigment abnormalities without additional features is caused by a mutation in the MLPH gene encoding melanophilin. A direct interaction exists between the SHD domain of melapholin and Rab27a and between the C-terminal domain of melanophilin and myosin Va [36]. Therefore, although the Rab27a/Mlph/MyoVa complex is crucial for the trafficking of melanosomes, neurodevelopmental abnormalities are specific for the Myo5a deficiency, and Rab27a uses a different effector for its function in the CTL.
Rab proteins in immunology
It has already been mentioned that GS type II patients with Rab27a mutations develop the hemophagocytic syndrome, which is characterized by episodes of life-threatening uncontrolled T lymphocyte and macrophage activation [17]. During these episodes, activated T cells and macrophages infiltrate various organs (including the brain), leading to tissue damage, organ failure and death in the absence of immunosuppressive therapy. In practice, only bone-marrow or cord blood stem cell transplantation can be curative for this condition [33].
The important function of Rab proteins in intracellular traffic, endocytosis and exocytosis explains their role in counteracting bacterial and viral infections. Phagocytosis by macrophages leads to the destruction of bacteria. Endocytosis leads to the formation of phagosomes, but phagosomes fuse with lysosomes (phagolysosomes), resulting in the release of toxic products that kill the bacterium as well as the release of the degradation products by exocytosis [19]. However, some bacteria survive this process and avoid bactericide by escaping from the phagosomes into the cytoplasm [19]. Listeria monocytogenes is a paramount example. It has been shown that the stimulation of macrophages with interferon (IFN)-γ, a cytokine that is secreted by natural killer and T helper cells, results in the expression of at least 200 proteins, including Rab5a [11]. In L. monocytogenes-infected macrophages, overexpression of Rab5a stimulates the intracellular degradation of the pathogen [6]. It has been suggested that Rab5a participates in the active recruitment of Rac2 to phagolysosomes, which is important for the destruction of the bacterium [27]. Other microbes develop antiphagocytosis (e.g. Yersinia enterocolitica) or inhibit the formation of phagosomes (e.g. Salmonella typhimurium). In Salmonella infections, there is evidence that Rab5a is stabilized on the phagosome by the Salmonella type III secretory protein SopE [23]. SopE has GEF activity for Rab5a, stimulating activated GTP-bound Rab5a and preventing its membrane extraction by RabGDI. A general conclusion that can be drawn is that parasites recruit endocytic Rabs to prevent or delay the formation of the degradative phagolysosome in the host organism.
Viruses enter host cells by receptor-mediated endocytosis, but they usually escape from endosomal vesicles to enter either the cytosol or the nucleus where they replicate. The binding of hepatitis C virus (HCV) non-structural protein (NS) with Rab1 GTPase-activating protein suggests that viruses are able to subvert host cell machinery in this way because this binding is necessary for HCV replication.
Rab1 depletion decreases HCV-RNA levels, which could lead to therapeutic applications [34].
Rab proteins in vision
Choroideremia is an X-linked disease that involves the degeneration of the retinal pigment epithelium and the adjacent choroid and retinal photoreceptor cell layers, leading to blindness. The mutated gene in the choroideremia is one of the two REP isoforms, REP1 [3, 30]. Although the other isoform, REP-2, seems to be sufficient for the geranylgeranylation of all Rab GTPases in all tissues, the retinal pigment epithelium seems to be the exception. A role for REP-2 in human disease has not yet been discovered. REP-1 is essential for the efficient geranylgeranylation of Rab27a in the retinal pigment epithelium. Thus, a lack of REP1 leads to a lack of functional Rab27a specifically in the retinal pigment epithelium [30, 31]. The degeneration of this epithelium and its adjacent layers may be due to deficient melanosome transport and, consequently, a lack of protection against harmful light exposure.
Rab proteins in neurology
A subgroup of patients with X-linked non-specific mental retardation have mutations in the GDI1 gene for one of the GDI isoforms, GDI-α [9]. This isoform is particularly abundant in the brain, and dysfunctional membrane recycling of one or more Rab GTPases in brain synapses, leading to aberrant neurotransmission, is likely to underlie the symptoms in this disease.
Hereditary sensory and autonomic neuropathies (HSAN), known for many years as familial dysautonomia in Jewish patients, are characterized by the loss of sensation for pain and temperature, alacrima, excessive sweating and the absence of fungiform tongue papillae. The onset is congenital, and the transmission is autosomal recessive. The clinical symptoms of HSAN have been classified into five types [21]. HSAN type II encompasses patients with Charcot–Marie–Tooth disease type II. This is an axonal neuropathy with a median motor conduction velocity of >38 m/s. This peripheral neuropathy starts in the second or third decade of life and presents as mixed motor and sensory involvement associated with ulcers. Four mutations in the Rab7 gene have been identified in four families with Charcot–Marie–Tooth disease type 2B (CMT2B), with symptoms leading to ulcerations and amputations [38, 39]. However, a hallmark feature of patients with HSAN type 1 is lancinating pain, which is not commonly observed in CMT2B. Moreover, HSAN type 1 is associated with a mutation in serine palmitoyl transferase (SPT), a rate-limiting step in the sphingo-lipid synthesis. This overlapping of the two syndromes is possibly explained by Rab7 being involved in both the endocytosis and transport of sphingolipids [39].
The importance of Rab proteins in brain development and function is even more emphasized by the recent discoveries of the involvement of Rab3-associated proteins in human disease [4, 5]. Rab3A is the most abundantly expressed protein in the brain, where it is present in all synapses and involved in calcium-dependent neurotransmitter release [13]. The activity of Rab3 proteins is tightly regulated by Rab3GAP, which specifically converts active Rab3-GTP to the inactive-GDP form. Germline-inactivating mutations in the catalytic subunit of Rab3GAP (Rab3GAP1) cause Warburg Micro syndrome, an autosomal recessive disorder characterized by ocular defects (microphthalmos, microcornea, congenital cataracts and optic atrophy), neurodevelopmental defects (microcephaly, cortical gyral abnormalities, such as pachygyria and polymicrogyria, hypoplasia of the corpus callosum, severe mental retardation, and spastic cerebral palsy) and hypothalamic hypogenitalism [4]. Mutations in the non-catalytic subunit of Rab3GAP (Rab3GAP2) have been described in patients with Martsolf syndrome, an autosomal recessive disorder with a similar but milder phenotype than the Warburg Micro syndrome, including congenital cataracts, hypogonadism and mild mental retardation [5]. The precise mechanisms whereby the Rab3GAP1 and Rab3GAP2 mutations cause the human phenotype is still unclear.
Rab proteins in endocrinology
Abnormal trafficking of the insulin-sensitive glucose transporter GLUT4 has been described in patients with diabetes type 2. Glucose transporter 4 accumulates in the dense membrane compartments, suggesting that defects in membrane trafficking may be involved in insulin resistance [12]. Rab4 has been found to be implicated in GLUT4 biogenesis, sorting and exocytosolic movement but more studies are needed to define the exact role of this and other Rabs and Rab-associated proteins in GLUT4 trafficking [16]. Rab4 mutations in humans or mice do not yet exist.
Rab27a is necessary for insulin secretion [2]. Diabetes mellitus is characterized by both low insulin and high glucagon levels. These conditions have been shown recently to be due to the expression of an inducible cAMP early repressor (ICER) [1], resulting in a repression of the transcription of the insulin gene, and associated with a diminished level of granuphilin, a Rab27a-dependent protein. However, patients with GS type II-homozygous deficiency in Rab27a do not have diabetes mellitus.
The control of thyroid hormone production depends on endocytic catalysts and the tandem regulators Rab5a and Rab7, which have been found in excess in thyroid adenomas [8].
Rab proteins in nephrology
Nephrogenic diabetes insipidus (NDI) is characterized by the inability to concentrate the urine in response to the antidiuretic hormone vasopressin (AVP). Under normal conditions, binding of AVP to the vasopressin receptor (V2R) leads to the insertion of aquaporin 2 (AQP2) water channels in the apical membrane of the renal collecting duct [28] (Fig. 2). The defect can be inherited and is either X-linked due to mutations in the genes encoding the V2R or autosomal recessive or dominant in mutations in the AQP2 gene. The trafficking of AQP2 in the cell and its internalization are cAMP and especially Rab11-dependent [40]. However, Rab11 mutations in humans or mice are not yet described.
Fig. 2Regulation of aquaporin-2 (AQP2)-mediated water transport by vasopressin (AVP). Shown is a nephron with a magnified principal cell. In this cell, the vasopressin V2 receptor (V2R), stimulatory GTP binding protein (Gs), adenylate cyclase (AC), ATP, cAMP, and phosphorylated proteins (O-P) are indicated. This figure has been used with permission of the American Journal of Physiology and Renal Physiology
Conclusion
The Rab GTPases are a large family of proteins with a variety of regulatory functions in membrane trafficking. The central role of these proteins has become clear during the past decade, as part of the progress that has been made in understanding in detail the mechanistic principles of transport vesicle formation, movement, and fusion. Sequencing of the human genome has enabled researchers to realize the diversity of the Rab gene family, although the functions of the majority of the gene products are still unknown. The availability of complete genomic sequences as well as the important advances in molecular and cell biological methods that have already been made hold the promise of significant progress being booked in our understanding of Rab function in the near future. The identification of further genes involved not only in monogenic but also in common multifactorial human vesicle-trafficking disorders will result in a better understanding of this complex transport pathway but, more importantly, will also lead to opportunities to develop novel treatments.


The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division.
Introduction
RNA polymerase II (pol II) transcribes the coding genes, and its activity is the main target for transcriptional control in all eukaryotes. The pol II enzyme consists of 12 subunits, which are highly conserved during evolution. The specific functions of the smaller subunits such as Rpb4 are relatively poorly understood, and they can show differences between organisms. For example, Rpb4 is essential in mammals and fission yeast (Schizosaccharomyces pombe), but not in budding yeast (Saccharomyces cerevisiae). Here we use fission yeast as a model to learn more about particular roles of Rpb4 in genome-wide transcription.
Much of our current understanding of the mechanism of transcription and the transcription machinery comes from studies using budding yeast. The distantly related fission yeast provides a valuable complementary model system to dissect the functions of different components of the transcriptional apparatus, because its transcription mechanism is more similar to that of mammals in some respects. For example, the initiation of transcription in both mammalian cells and S. pombe occur ∼30 bp downstream of the TATA box, whereas in S. cerevisiae this distance can vary between 40 and 120 bp (Li et al. 1994). Moreover, transcriptional initiation from mammalian promoters introduced into S. pombe occurs at the same site as in mammalian cells (Toyama and Okayama 1990).
As in other eukaryotes, S. pombe pol II is a multi-subunit enzyme containing the 12 subunits Rpb1 to Rpb12. The four core subunits, Rpb1, Rpb2, Rpb3 and Rpb11, are homologous to the β′, β, α′ and α subunits, respectively, of the eubacterial RNA polymerase. Five subunits, Rpb5, 6, 8, 10 and 12, are shared by the three eukaryotic RNA polymerases I, II and III, whereas Rpb4, Rpb7 and Rpb9 are subunits specific to pol II (Mitsuzawa and Ishihama 2004). The functions of most of these smaller subunits are not well characterized.
Rpb4 and Rpb7 form a conserved heterodimer complex in archaebacteria, yeast, plants and humans (Choder 2004). Crystallographic studies of pol II and biochemical data provide evidence for a role of the Rpb4/7 heterodimer in transcription initiation and RNA binding in S. cerevisiae (Armache et al. 2005; Bushnell and Kornberg 2003; Edwards et al. 1991; Orlicky et al. 2001). S. pombe Rpb7 interacts with an RNA-binding protein, implying a role in the coupling of RNA processing to transcription, and it associates with glyceraldehyde-3-phosphate dehydrogenase and actin (Mitsuzawa et al. 2003, 2005).
The in vivo functions of Rpb4 have been characterized in S. cerevisiae, and several lines of evidence suggest that it plays an important role in the response and survival to stress (reviewed by Choder 2004). S. cerevisiae Rpb4 is also important for activated transcription from a subset of promoters and in carbon and energy metabolism at moderate temperatures (Pillai et al. 2001, 2003). Recent data show that it is required for the decay of a class of mRNAs whose products are involved in protein synthesis, and it interacts with subunits of the mRNA decay complex Pat1/Lsm1-7 that enhances decapping (Lotan et al. 2005).
We are only beginning to understand the role of the Rpb4 subunit in S. pombe: it binds to the TFIIF-interacting carboxyl-terminal domain (CTD) phosphatase Fcp1, and it has been proposed to play a role in the assembly of the Fcp1–pol II complex, thereby promoting CTD phosphorylation for the reutilization of pol II in a new cycle of transcription (Kimura et al. 2002). S. pombe Rpb4 exhibits features that resemble its budding yeast counterpart, while others place it closer to its orthologs in multicellular eukaryotes. It is essential for viability, whereas in S. cerevisiae it is not required for cell growth under optimal conditions (Sakurai et al. 1999; Choder 2004). S. pombe Rpb4 contains 135 amino acids, placing it closer in size to its counterparts in humans (142 amino acids) and plants (138 amino acids) than to S. cerevisiae (221 amino acids). Stoichiometric amounts of Rpb4 associate with pol II in S. pombe and higher eukaryotes, while the fraction of pol II molecules containing Rpb4 in optimally growing S. cerevisiae cells is only about 20%. Finally, Rpb4 is more tightly associated with S. pombe pol II than with S. cerevisiae pol II (Sakurai et al. 1999).
Here, we provide evidence that Rpb4 influences the growth and gene expression of S. pombe cells in a dose-dependent fashion under optimal conditions. We show that lower levels of rpb4 expression do not affect the survival of cells under several stress conditions. Our results imply a particularly important role for Rpb4 in the expression and regulation of a subset of genes involved in cell separation. The defects in cell growth and separation as well as the gene expression signatures associated with low levels of rpb4 expression are similar to the phenotypes and expression signatures of a range of transcriptional regulatory mutants with defects in cell separation.
Materials and methods
Strains and molecular genetic methods
The S. pombe strains used in this study are listed in Table 1. JB22 was the parental haploid strain used to generate the strains expressing rpb4 under the P3nmt1, P41nmt1 and P81nmt1 promoters (Basi et al. 1993). The strains were constructed by PCR using the pFA6a-kanMX6-P3/P41/P81nmt1 constructs as described (Bähler et al. 1998), and the kanamycin-resistant transformants were selected on KsNoT media (Linder et al. 2002). Recombinant DNA methods were performed as described (Sambrook et al. 1989) and standard methodology and media for the manipulation of S. pombe were used (Moreno et al. 1991).
Table 1Strains used in this studyStrainRelevant genotypeSourceJB22972 h−Leupold (1970)JB394 kanMX6-p3nmt1-rpb4 h−This studyJB395KanMX6-p41nmt1-rpb4 h−This studyJB396kanMX6-p81nmt1-rpb4 h−This study
Growth experiments
Pre-cultures for the growth curves were made by inoculating the respective strains in Edinburgh Minimal Medium (EMM) without thiamine or with 15 μM thiamine and growing the cultures for ∼17 h at 32°C with shaking. Subsequently, the pre-culture was diluted into 50 ml of fresh EMM medium at an OD600 = 0.05–0.1 and allowed to grow at 32°C with shaking. Samples were removed at various timepoints thereafter for measuring ODs at 600 nm, microscopic examination, and microarray hybridization experiments. The low OD measurements in cells expressing rpb4 under the P41nmt1 and P81nmt1 promoters is not caused by the phenotype, because mutants with similar phenotypes such as sep1 show strong increases in OD during growth (unpublished data).
Quantitative RT-PCR experiments
To measure rpb4 expression, the strains expressing rpb4 under the three promoters were grown for 24 h in EMM medium at 32°C (promoter on) or cultivated for a further 21 h in the presence of 15 μM thiamine (promoter off). RNA was isolated by phenol extraction, purified using the RNeasy kit (Qiagen), and treated with RNase-free DNase (Roche). The reverse transcription reactions were performed using Superscript III (Invitrogen). Real-time PCR was performed using the Brilliant® SYBR® Green QPCR Core Reagent Kit (Stratagene) on an Mx3000P QPCR system (Stratagene) with primers specific for rpb4 or fba1 (control) (sequences available on request). Arbitrary expression units were calculated using a standard curve made from serial dilutions of fission yeast genomic DNA.
Stress response assays
Serial dilutions (1/10) of rpb4 strains generated in our study were spotted on EMM plates with or without 15 μM thiamine. The plates were supplemented with 0.5 mM hydrogen peroxide or different concentrations of sorbitol (1, 2, 3 and 4 M) to test the response to oxidative and osmotic stress, respectively. These plates were incubated at 32°C for 2 days and photographed. The effect of temperature stress was tested by incubating the strains at 36°C for 2 days.
Microscopy
Unfixed cells were observed at 2-h intervals for 25 h by light microscopy using a Zeiss Axioscope fluorescence microscope set up for differential interference contrast (DIC) with a 40× objective and Axiovision digital imaging system. To stain the nuclei, cells were spread onto microscopic glass slides. Subsequently, the cells were fixed by heating at 70°C for 1 min and DAPI (4′,6′ diamidino-2-phenylindole) was added at a final concentration of 1 μg/ml. To view the division septa, calcofluor was added to cultures at a final concentration of 5 mg/ml and the cultures were incubated in the dark at room temperature for 5 min. The cells were washed twice with phosphate buffer saline, and 5 μl of cell suspension was spotted onto microscopic glass slides. DAPI- and calcofluor-treated cells were visualized under a Zeiss Axioscope microscope as above.
Microarray experiments
To compare the gene expression profiles as a function of rpb4 expression, cells were grown in the presence of thiamine to OD600 of 0.2, and 50 ml of culture was centrifuged. The remaining culture was washed four to five times with EMM and subsequently grown in EMM medium without thiamine for 17 h and centrifuged. To identify transcripts affected by lowered rpb4 expression, the desired strains were grown in the presence of thiamine for 21 and 23 h and harvested. A wild-type parental strain grown in the presence of thiamine served as a reference. Although thiamine leads only to minimal expression changes (Jenkins et al. 2005), this experimental design corrects for effects caused by the presence of thiamine. Pellets were frozen in liquid nitrogen and kept at −70°C until further processing. RNA was isolated by phenol extraction and purified by the RNeasy kit (Qiagen). cDNA probes were prepared with Superscript (Invitrogen) and labeled with Cy3 or Cy5. Detailed methods were as described (Lyne et al. 2003; http://www.sanger.ac.uk/PostGenomics/S_pombe). Microarrays were scanned with a Genepix 4000B scanner and analyzed with Genepix software (Axon Instruments). Data filtering, normalization and quality control were performed as described (Lyne et al. 2003). The data are based on two to three independent biological repeats with dye swaps. To quantify global effects on gene expression, we repeated microarray analysis spiked with external control RNAs. The expression data were normalized using Bacillus subtilis spikes added in equal quantities to the RNA samples before labeling (Lee et al. 2005). Each microarray contains 720 control elements for the spikes, which are spread across the complete grid of the array in a spatially even manner. Clustering and visualization were done with GeneSpring. Gene annotations were taken from the S. pombe GeneDB database (www.genedb.org/genedb/pombe/index.jsp). Overlaps between different gene lists were calculated using the hypergeometric distribution. All normalized data sets are available from our website: http://www.sanger.ac.uk/PostGenomics/S_pombe
Results
Dosage-dependent effect of Rpb4 on cell growth
Rpb4 is an essential subunit of S. pombe pol II. To elucidate its in vivo function(s), we constructed haploid strains expressing rpb4 from its normal chromosomal location using the thiamine-regulated nmt1 promoter. Three different versions of this promoter were used: the wild-type P3nmt1 promoter has the highest level of promoter activity and maintains substantial basal levels of expression even under repressed conditions; the two derivatives, P41nmt1 (medium strength) and P81nmt1 (weakest strength) have reduced levels of activity due to mutations in the TATA box (Basi et al. 1993).
We performed quantitative PCR of cells expressing rpb4 under the three promoters, both in the absence (nmt1 promoter ‘on’) or presence (nmt1 promoter ‘off’) of thiamine to verify changing levels of rpb4 mRNA (Fig. 1). Expression levels of rpb4 showed a range of more than 1,000-fold in the different conditions, and the relative expression levels were as expected for the three promoters of different strength. The rpb4 levels were especially low when expressed from the P41nmt1 and P81nmt1 promoters in the presence of thiamine. The fba1 gene is highly expressed and was used as a control gene. Although it shows much less variation in expression levels than rpb4, there are some changes in accordance with rpb4 levels (Fig. 1). This probably reflects effects on global transcription as a function of rpb4 expression levels (see below).
Fig. 1Expression levels of rpb4 mRNA when transcribed from different promoter constructs. Cells expressing rpb4 from the P3nmt1, P41nmt1 or P81nmt1 promoter were grown for 24 h in EMM medium at 32°C (promoter on; gray bars) or cultivated for a further 21 h in the presence of thiamine (promoter off; white bars). rpb4 (left) and fba1 (right; control) mRNA levels from the same cells were measured by quantitative RT-PCR. The PCR was performed on reverse transcription reactions, which were carried out in the presence (RT+) or absence (RT−) of reverse transcriptase
To analyze effects of rpb4 expression levels, the three strains were grown at 32°C either in the absence or presence of thiamine, and cell growth was monitored. In the absence of thiamine, all strains grew similar to each other and to the wild-type strain (Fig. 2a). This shows that rpb4 expressed from the weak P81nmt1 promoter under induced conditions was sufficient to sustain normal cell growth. When these strains were grown in the presence of thiamine, however, there was a direct correlation between residual promoter activity and growth rate: cells expressing rpb4 from the P3nmt1 promoter showed the same growth rate as wild-type cells, while cells expressing rpb4 from the P41nmt1 promoter and, even more so, from the P81nmt1 promoter showed reduced growth rates (Fig. 2b). Thus, in the presence of thiamine, the lowered Rpb4 levels become limiting for cell growth, and residual growth directly reflects basal expression levels from the different promoter constructs.
Fig. 2Effect of Rpb4 on cell growth. Wild-type cells (filled circle) and cells expressing rpb4 from either the P3nmt1 (filled square), P41nmt1 (filled triangle) or P81nmt1 (crosses) promoter were grown in EMM medium at 32°C. OD600 was measured at the indicated times. a In the absence of thiamine, all strains grew at similar rates. b In the presence of thiamine, the growth rates were dependent on the residual levels of rpb4 being expressed from the different nmt1 promoters
Effect of Rpb4 on cell growth under stress conditions
Rpb4 is required for stress response in S. cerevisiae (Choder 2004). We therefore, determined if Rpb4 played a similar role under stress conditions in S. pombe. Cells expressing varying levels of rpb4 were subjected to temperature stress (36°C), osmotic stress (1 M sorbitol) or oxidative stress (0.5 mM hydrogen peroxide). We found that neither in the presence nor absence of thiamine, the cells exhibited any increased stress sensitivity compared to control cells when grown under the different stress conditions (data not shown). Similar results were obtained when cells were grown in up to 2 M sorbitol. No significant growth of either the wild-type or Rpb4 expressing cells was observed in the presence or absence of thiamine at 3 and 4 M concentrations of sorbitol. These observations indicate that low levels of Rpb4 do not affect the survival of cells under any of the tested stress conditions.
Repression of rpb4 transcription causes defects in cell separation
We next examined whether different levels of rpb4 expression would influence specific cellular processes. Cells expressing rpb4 from any of the three nmt1 promoters in the absence of thiamine did not lead to any obvious phenotypes even after 25 h of growth in medium without thiamine (Fig. 3a).
Fig. 3Rpb4 affects cell separation and morphology. Cells expressing rpb4 from either the P3nmt1 or P81nmt1 promoter were analyzed by microscopy. a Cells grown in EMM in the absence of thiamine (promoter ‘on’) at 32°C appear like normal, wild-type cells. b When grown in the presence of thiamine (promoter ‘off’), cells expressing rpb4 from the P3nmt1 promoter appear normal, while those expressing rpb4 under the P81nmt1 promoter display defects in cell morphology and septation. Multiple phenotypes including elongated cells with single or multiple septa and growth by branching are evident. c Cells expressing rpb4 under the P81nmt1 promoter were analyzed by fluorescence microscopy in the presence of thiamine. DAPI staining revealed single nuclei in each cell compartment, while calcofluor staining showed septa (some of them aberrant) separating the cell compartments. d Control cells and mutants showing similar phenotypes as cells expressing rpb4 under the P81nmt1 promoter cells. From left to right: wild-type cells, sep1 deletion mutants, ace2 deletion mutants and sep1 ace2 double deletion mutants
In thiamine-containing medium, however, cells expressing rpb4 from the weak P81nmt1 promoter became more elongated than the wild-type cells after 19 h of growth (Fig. 3b). Moreover, after continued incubation in the same medium for another 2 h (21 h with thiamine), cells also showed defects in cell separation, which became more evident after an additional 4 h of incubation (25 h with thiamine) (Fig. 3b). Most cells were elongated with either a single or multiple septa, and some cells also displayed bent morphologies and/or growth by branching. Cells expressing rpb4 from the medium strength P41nmt1 promoter displayed no obvious cell separation defects until 23 h of growth in the presence of thiamine. Similar defects as described above, however, became apparent after 23 h of growth in thiamine (data not shown). In contrast, cells expressing rpb4 from the strong P3nmt1 promoter did not exhibit any aberrations in morphology or division after growth in thiamine-containing medium for 25 h (Fig. 3b) and continued to look like wild-type cells even at 40 h of growth in thiamine-containing medium (data not shown). Thus, the basal level of expression from the strongest promoter is sufficient to prevent the cell separation defects seen with the weaker promoters.
We investigated these phenotypes in more detail for cells expressing rpb4 under the P81nmt1 promoter by staining the division septa and cell nuclei (Fig. 3c). Several nuclei were present separated from each other by either a single septum or multiple, aberrant septa. Some cells also contained large deposits of septal material at random positions (Fig. 3c). We conclude that lowered rpb4 expression levels lead to filamentous growth with multiple cells remaining attached to each other. Similar phenotypes to those of cells expressing rpb4 under the P81nmt1 promoter have observed in several different mutants (see Discussion); for example, deletions in two transcription factor genes, sep1 and ace2, also lead to defects in cell separation (Fig. 3d; Bähler 2005). These findings raise the possibility that Rpb4 plays a specific role in cell separation after cytokinesis.
Low levels of rpb4 transcripts most strongly affect expression of genes involved in septation
We used S. pombe whole-genome microarrays to investigate the effects on gene expression caused by low levels of rpb4. Wild-type cells and cells expressing rpb4 under the different regulatable promoters were grown in the presence and absence of thiamine followed by RNA extraction. Cells expressing rpb4 under the P3nmt1 promoter did not show any significant differences in gene expression profiles when compared with wild-type cells, either in the presence or absence of thiamine (data not shown), consistent with our earlier observations that these cells show normal growth and phenotypes. If rpb4 was expressed under the weakest promoter in the presence of thiamine, however, gene expression changed substantially. As down-regulation of rpb4 is expected to have a global effect on transcription, we used external spikes for normalization (Materials and methods). This revealed that mean transcript levels were 26–36% lower compared to wild-type cells grown under the same conditions (Fig. 4a). The expression levels of rpb4 were two- to threefold lower under these conditions compared to the levels under its own promoter. Thus, lowered rpb4 expression leads to reduced overall transcript levels.
Fig. 4Effects on global and specific gene expression as a function of rpb4 levels. a Microarray experiments of cells expressing rpb4 under the weakest promoter. The histograms show the distribution of gene expression ratios of P81nmt1-rpb4 cells grown in the presence of thiamine for 21 and 23 h relative to wild-type cells grown in the presence of thiamine. Genes in both histograms are colored by their relative expression levels at 21 h as indicated at upper right. External spikes have been used for normalization, revealing a global down-regulation of transcript levels by 26% (21 h) to 36% (23 h) on average when rbp4 expression is lowered. The averages of two independent repeats for each timepoint are shown. b Overlap between the genes downregulated when rpb4 is expressed under the weakest promoter in the presence of thiamine and genes downregulated in both sep10 and sep15 mutants (Lee et al. 2005). The number in brackets represents the overlap expected by chance, given the sizes of the gene sets considered and the total number of 5,180 genes. The overlap is highly significant as calculated using the hypergeometric distribution (P ∼ 1 × 10−30). c Hierarchical cluster analysis of 249 genes that are either 1.5-fold upregulated or threefold downregulated when rbp4 is expressed under the weakest promoter in presence of thiamine. Horizontal rows represent the profiles of hierarchically clustered genes. Columns represent experiments of different mutant strains and different time-points for the same strain. The transcript levels for each strain (indicated at bottom) relative to levels in wild-type cells are color-coded as indicated at right, and missing data are shown in gray. The microarray data from mcs6, pmh1, sep10 and sep15 mutants are from Lee et al. (2005), and the data from sep1 and ace2 mutants are from Rustici et al. (2004)
On top of this genome-wide down-regulation of transcription, 96 transcripts were at least 1.5-fold upregulated in three of the four samples tested, while 150 genes were at least threefold downregulated in three of the four samples tested upon lowering rpb4 expression (Supplementary Tables S1 and S2). These genes were categorized according to their functions (Supplementary Table S3). The majority of the genes whose expression levels were affected by rpb4, encoded proteins involved in metabolism and transport. Many of the upregulated transcripts in cells with lowered rpb4 expression levels overlapped with genes upregulated during different environmental stresses (Chen et al. 2003) and nitrogen starvation (Mata et al. 2002) (P ∼ 1 × 10−13 to 1 × 10−36). This probably reflects an indirect effect caused by the defects in these cells. A substantial number of the downregulated genes in the category of cell cycle and cell division played specific roles in cytokinesis and cell separation. For example, the two genes eng1 and agn1 encode β-glucanase and α-glucanase, respectively; Eng1 degrades the primary division septum between the new ends of daughter cells and Agn1 hydrolyzes the old cell wall surrounding the septum leading to full separation of daughter cells (Martín-Cuadrado et al. 2003; Dekker et al. 2004). The genes adg1 and adg3 have also been shown to be involved in cell separation (Alonso-Nunez et al. 2005), and rng3 encodes a protein involved in formation of contractile ring during cytokinesis (Wong et al. 2000).
Both the lists of genes being upregulated and downregulated after rpb4 down-regulation showed highly significant overlaps with lists of genes upregulated or downregulated, respectively, in sep10, sep15, mcs6 and pmh1 mutants (P ∼ 1 × 10−16 to 1 × 10−47) (Lee et al. 2005), whose genes encode different proteins involved in transcriptional regulation (see Discussion). Among the downregulated genes, the most pronounced overlap was observed with sep10 and sep15 mutants (Fig. 4b). The downregulated genes also showed some overlap with genes downregulated in sep1 and ace2 mutants (P ∼ 1 × 10−14) (Rustici et al. 2004), which encode cell-cycle transcription factors. Figure 4c shows a cluster analysis of the genes most strongly regulated by rpb4 repression and their expression levels in the various transcriptional mutant backgrounds. The sep10, sep15, mcs6 and pmh1 mutants show highly similar expression signatures to cells with compromised rpb4 expression, while the overlap with sep1 and ace2 mutants is less evident. Intriguingly, if rpb4 was overexpressed using the medium strength promoter, the expression levels of these genes were reversed, with downregulated genes becoming upregulated and upregulated genes becoming downregulated (Fig. 4c). Thus, transcript levels of this gene set are particularly responsive to the transcript levels of rpb4.
Discussion
We have analyzed functions of the Rpb4 subunit of pol II in S. pombe. Despite the overall conservation in structure and function of pol II across species, the Rpb4 subunit presents a special case. S. pombe Rpb4 is similar to its orthologs in higher eukaryotes with respect to its structure and essential nature (Sakurai et al. 1999), but in contrast to its human ortholog, it can form stable hybrid dimers with S. cerevisiae Rpb7 (Sakurai et al. 1999) and can rescue the temperature-sensitive phenotype associated with the lack of S. cerevisiae Rpb4 (Shpakovski et al. 2000). Thus, S. pombe Rpb4 may have intermediate characteristics useful to bridge the knowledge from S. cerevisiae and multicellular eukaryotes.
We used regulatable gene expression constructs to study the in vivo roles of Rpb4. Under optimal conditions, rpb4 influences the growth of cells in a dose-dependent manner when expression drops below a certain level using the regulatable promoters (Figs. 1, 2). In S. cerevisiae, deletion of the RPB4 gene leads to slow cell growth under optimal conditions (Woychik and Young 1989; Choder 2004), while in S. pombe rpb4 is essential (Sakurai et al. 1999). We found that low levels of rpb4 expressed from the weakest promoter were sufficient for the cells to survive a range of different stress conditions including temperature stress. Accordingly, core stress genes were actually upregulated in rpb4 mutants, and S. pombe Rpb4 is therefore unlikely to play any specialized role during environmental stress. S. cerevisiae Rpb4 is dispensable for oxidative and osmotic stress (Maillet et al. 1999), but unlike in S. pombe, it is required for survival of temperature stress (Woychik and Young 1989).
Fission yeast cells grown under low expression of rpb4 were elongated and showed defects in cell separation as indicated by the accumulation of division septa, some of them highly aberrant (Fig. 3). Strains with cell separation defects similar to the ones described here include mutants in components of the exocyst complex (sec6, sec8, sec10 and exo70), an anillin homolog (mid2), septins (spn3, spn4), an endo-α-1,3-glucanase (agn1), an endo-β-1,3-glucanase (eng1), calcineurin (ppb1), a MAP kinase and phosphatase (pmk1, pmp1), two transcription factors (sep1, ace2), and subunits of the mediator complex (sep10, sep11 and sep15) (Sipiczki et al.1993; Yoshida et al. 1994; Longtine et al. 1996; Toda et al.1996; Sugiura et al. 1998; Grallert et al. 1999; Wang et al. 2002; Martín-Cuadrado et al. 2003; Spåhr et al. 2003; Tasto et al. 2003; Dekker et al. 2004). In S. cerevisiae, haploid rpb4 deletion mutants exhibit an axial budding pattern with normal yeast cell morphology, but diploid rpb4 mutants are more elongated than wild-type cells during nitrogen starvation and grow in a unipolar pattern, resembling pseudohyphae formation (Pillai et al.2003).
Lowered expression of rpb4 causes relatively modest reductions in global transcription, which probably leads to the reduced growth rates under these conditions (Fig. 4a). In addition, low rpb4 transcription results in substantially lowered expression of a distinct subset of genes. Among the strongly affected genes were eng1, agn1, adg1 and adg3 with known functions in cell separation, which are targets of a transcriptional cascade regulated by the Sep1 and Ace2 transcription factors (Rustici et al. 2004; Alonso-Nunez et al. 2005). Accordingly, the genes affected by lowered rpb4 expression significantly overlapped with genes affected in sep1 and ace2 mutants. Much stronger similarities, however, were evident with the gene expression signatures of several strains mutated in the sep10, sep15, mcs6 and pmh1 genes, which encode various components of the transcriptional machinery. Sep10 and Sep15 are essential components of the mediator complex of pol II, while Mcs6 and Pmh1 are components of a complex homologous to metazoan Cdk7-cyclinH-Mat1, a cyclin-dependent kinase-activating kinase (CAK) and a pol II CTD kinase associated with transcription factor IIH (Spåhr et al. 2001, 2003; Lee et al. 2005). The overlap in expression signatures between these four mutants and cells with lowered rpb4 expression was evident for both upregulated and downregulated genes. These differentially regulated genes showed reverse regulation (upregulated genes became downregulated and vice versa) when rpb4 was overexpressed from the medium strength promoter (Fig. 4c). Thus, the expression of this gene set is particularly responsive to rpb4 expression levels and also depends on a range of other factors involved in general transcription. This could either reflect a specific transcriptional pathway involving Rpb4 and the other factors (either directly or indirectly via the control of a regulator), or the affected genes could be generally sensitive to transcriptional interference. In any case, these data show that the transcriptional pathway required for cell separation in fission yeast (Bähler 2005) is strongly affected when interfering with rpb4 expression levels.
We speculate that the Rpb4/Rpb7 complex is involved in linking the transcriptional output to the metabolic status of the cell. The rbp4 and rpb7 transcripts are down-regulated when cells enter stationary phase (unpublished observations), Rpb7 interacts with the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Mitsuzawa et al. 2005), and global transcription is sensitive to rpb4 expression levels. It is possible that the transcriptional pathway required for cell separation is particularly sensitive for compromised transcription to adjust the growth mode to availability of nutrients: in abundant nutrients, global transcription is efficient and growth is best as single cells, while in limiting nutrients, global transcription and cell separation are compromised, and cells grow as multicellular pseudohyphae, which may allow a more efficient grazing for new nutrients as growth is directed. In the first description of S. pombe, multi-cellular pseudohyphae similar to those seen in rpb4 mutants have been observed (Lindner 1893); the natural capacity to switch to multi-cellular growth might have been lost in the yeast strains cultured in the laboratory.
Taken together our data suggest that S. pombe Rpb4, besides its general role in transcription, performs a more specialized function in regulating a specific gene expression program required for cell separation at the end of the cell cycle. It is possible that this role is carried out by interacting with other general factors of the transcriptional machinery such as the mediator and CTD kinase as suggested by similarities in phenotypes and expression signatures in our study. Accordingly, a comparison of the X-ray crystallographic structure of the complete pol II, including the Rpb4/7 heterodimer, with an electron microscopic structure of the pol II-mediator complex, has indicated that Rpb4/7 may interact with the mediator (Bushnell and Kornberg 2003). Alternatively, the subset of genes affected by changes in rpb4 expression (and by manipulating other general transcription factors) could reflect genes that are especially sensitive to transcriptional stress. This interpretation does not imply any specific gene expression program, although it could be used by the cell to adjust transcription to available nutrients. Given the conservation of Rpb4, it will be interesting to see whether the human ortholog plays a similar specialized role to transcribe particular subsets of genes.
Electronic supplementary material
Supplementary material

transcription
growth
cell division
s. pombe