Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target.
Introduction
The murine double minute (MDM) family members are key regulators of levels and activity of the oncosuppressor p53 [1]. MDM2, acting as specific E3 ubiquitin ligase and as transcriptional repressor, is the best known among them. MDM2 overexpression has been observed in many human tumors characterized by tumor protein 53 (TP53) wild-type (wt) status, supporting the model of multiple means of p53 inactivation in tumor cells [2]. In recent years, another member of the MDM family, MDM4 (also named MDMX) has come into the limelight and its function in the inactivation of p53 has been revealed by molecular and genetic approaches [1, 3]. In particular, MDM4 can negatively regulate p53-mediated cell cycle arrest, a role that distinguishes its function from that of MDM2, more related to the suppression of p53-mediated apoptotic response [1, 3–5]. However, others have reported on the antiproliferative and proapoptotic effect of MDM4 in the presence of wild-type TP53 under conditions such as stress [6, 7]. Studies aimed at characterizing the human MDM4 (also named HDMX) status in human tumors have shown amplification of its locus in the presence of wild-type TP53 in breast cancers, glioblastoma, retinoblastoma, and soft-tissue sarcomas [8–11], confirming that it may contribute to p53 inactivation during tumorigenesis. The impact of human MDM4 on tumor properties as well as its behavior during tumor progression however still poorly explored.
In addition to the full-length (fl) protein, different splice variants of MDM4 have been described, two of which detected in human tumors. A short form named MDM4-S (a.k.a. HDMX-S) was identified first [12]. This variant derives from an alternative splicing that produces a truncated protein, containing essentially the p53-binding domain. It is a more potent inhibitor of p53 transcriptional activity than wt MDM4 and although present in both normal and tumor tissues, it is overexpressed specifically in tumor samples [11, 12]. Recently, our group has characterized another MDM4-splicing form, HDMX211 (hereafter named MDM4-211), in a thyroid tumor cell line and in some lung cancers [13]. This form derives from an aberrant splicing and produces a protein containing essentially the RING finger COOH-terminus, where the MDM2 binding site resides. As a consequence, this variant is able to stabilize MDM2 protein levels, thus contributing to inactivation of p53. These data, while underlying the complexity of MDM4 expression in human tumors, strongly encourage studies on the comparative analysis of this protein and its derivative forms in human cancer.
Thyroid tumors represent over 90% of all endocrine cancers and are characterized by different genetic alterations, among which TP53 mutations are confined quite exclusively to the infrequent anaplastic and poorly differentiated histotype [14]. According to current models of human carcinogenesis that consider inactivation of the oncosuppressor p53, a common feature of almost all tumors, MDM2 amplification and/or overexpression have been reported in thyroid tumors too [15–17]. However, the overall frequency of MDM2 overexpression is only about 30%, suggesting the existence of other pathways of p53 inactivation.
The aim of our study has been to analyze papillary thyroid carcinomas (PTC), the most frequent thyroid cancer, for the presence of MDM4 and its derivative forms to investigate alterations of these proteins in this tumor histotype and correlate them with histopathological features. In addition, we have analyzed MDM2 levels to assess the potential relationship between alterations of the two MDM family members. Our data have revealed the aberrant presence of variants MDM4-S and MDM4-211 messenger RNA (mRNA) in PTC as well as lack of correlation between MDM4 and MDM2 mRNA levels. Of note, levels of MDM4 mRNA were significantly downregulated in tumor samples in comparison to normal counterparts, and such downregulation appears significantly associated with tumor stage.
Materials and methods
Tissue samples and patients
Fifty-seven papillary thyroid carcinomas and 57 matched normal thyroid tissue samples from the contra-lateral lobe (CTRL) of 57 patients were studied. In addition, to confirm statistical analyses, 26 papillary thyroid carcinomas and three normal thyroid tissues from independent individuals were analyzed. All specimens were obtained from patients undergoing surgery at the University of Perugia from 1997 to 2007. Before the surgical procedure, all patients signed informed consent forms for collection of fresh thyroid samples for genetic studies. All specimens were sampled from the primary tumor at the time of surgery, snap frozen, and stored at −80°C until use. Tumors containing at least 70% of tumor cells based on the hematoxylin–eosin staining were selected. All normal thyroid tissue from the contra-lateral lobe were histopathologically analyzed for the presence of tumor. Available medical records of the patients were consulted to gain information about the clinical features of the disease and, when possible, the tumor stage was defined according to the sixth edition of the “American Joint Committee on Cancer” Cancer Staging Manual (American Joint Committee on Cancer 2002) based on pathological tumor–node–metastasis parameters and distinguishing patients in two groups: (1) patient age < 45 years (stage I and II), (2) patient age ≥ 45 years (stage I, II, III, IV).
The total of 83 papillary thyroid carcinomas included 55 of the classic variant, 20 of the follicular variant, five of other variants (tall cell, solid, diffuse sclerosing), and three showing the coexistence of PTC histology with areas of dedifferentiation. The mean period of follow-up was 45.8 ± 30.4 months.
Genetic analysis of TP53 mutation, BRAF mutations and ret/PTC rearrangements
Analysis of the TP53 gene status was performed as follows: exons 5, 6, 7, and 8 of p53 gene were sequentially amplified by polymerase chain reaction (PCR) assay with the use of AmpliTaq Gold (Applied Biosystems) and following primer sets: exon 5 sense: TTCCTCTTCCTACAGTACTC; exon 5 antisense: GCCCCAGCTGTTCAC; exon 6 sense: ACTGATTGCTCTTAG; exon 6 antisense: AGTTGCAAACCAGAC; exon 7 sense: AGTTGTGTTATCTCCTAG; exon 7 antisense: CAAGTGGCTCCTGAC; exon 8 sense: TCCTATCCTGAGTAG; exon 8 antisense: GTCCTGCTTGCTTAC. Purified PCR products were sequenced in both directions with the use of the BigDye terminator Cycle sequencing Kit (version 1.1, Applied Biosystems) and an ABI Genetic Analyzer (Model 3130, Applied Biosystems). Sequence data were analyzed by means of SeqScape software (version 2.1, Applied Biosystems) followed by manual review.
Search for mutations of BRAF was conducted by single-stranded conformational polymorphism (SSCP) screening of real-time (RT)-PCR products of exons 15, followed by sequencing, as previously described [18]. Screening for ret/PTC 1 and ret/PTC 3 rearrangements was performed by RT-PCR using primers spanning the breakpoints, as previously described [18].
Quantitative real-time PCR
Total RNA was extracted with Trizol™ (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer’s instructions. The expression of MDM2, MDM4, and MDM4-211 in tumors and matching normal thyroid samples was measured by quantitative real-time PCR (qRT-PCR), based on TaqMan methodology, using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) according to Giglio et al. [13]. Briefly, custom-designed TaqMan primers and probes (Applied Biosystems) specific for MDM4, MDM2, and MDM4-211 were used. Our results are expressed as relative units of target mRNA, referred to a sample called calibrator, chosen to represent 1× expression of the target gene. The calibrator used was the lowest value in the tissue collection under study. All analyzed samples express n-fold mRNA relative to the calibrator. Each sample mRNA was normalized relative to the β-actin mRNA. Predesigned TaqMan primers and probe (Applied Biosystems) for the housekeeping gene ß-actin were used. Each sample was treated with deoxyribonuclease (DNase) I amplification grade (Invitrogen) and tested before and after DNase treatment.
Comparative analysis of flMDM4 and MDM4-S was driven by qRT-PCR according to Bartel et al., [11] using specific probes and SYBR Master mix (Applied Biosystems) with evaluation of dissociation curves. A MDM4-S-to-flMDM4 ratio of >1.5 was considered indicative of overexpressed MDM4-S.
Western blot analysis
Tumor and normal tissue pairs whose material was sufficient for western blot analysis were selected. All samples were homogenized with Tissue Lyser (Qiagen) and western blots were performed according to Giglio et al. [13]. Briefly, samples were lysed in radio-immunoprecipitation assay buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% Na desoxicholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid) supplemented with a cocktail of protease inhibitors (Boehringer). Whole lysates were boiled in SDS Laemnli sample buffer, resolved by SDS-polyacrylamide gel electrophoresis on precast 10% gels (Invitrogen) and subsequently transferred to polyvinylidene fluoride membranes (Millipore). After protein transfer, membranes were blocked for 30 min with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat dry milk and incubated with specific antibodies. The following primary antibodies were used: rabbit anti-MDM4 polyclonal antibody R1 (raised against full-length MDM4 protein) and (Bethyl Laboratories) mouse anti-MDM2 monoclonal antibody 2A10 and monoclonal antibody Ab-1 (oncogene), mouse anti-α-tubulin monoclonal antibody DM-1A (Sigma), and mouse α-actin monoclonal antibody AC-40 (Sigma). MDM4 was probed with both Bethyl and R1 antibodies, giving similar results although, in comparison, the polyclonal antibody R1 was more sensitive. The homemade R1 antibody has been raised using the entire MDM4 protein. Experiments of epitope mapping by using MDM4-deletion mutants have shown that R1 does not recognize the NH2-terminus of MDM4. It is therefore unable to recognize the MDM4-S form.
Statistical analysis
Statistic analysis was carried out using the Analyze-it software for Microsoft Excel (Analyze-it Software, Ltd.). Spearman rank correlations and Kendall rank correlation (for measure of linear association) were used to evaluate the correlation between not-normally-distributed variables. To compare groups, we used the Wilcoxon Signed-Ranks test for related samples (57 tumors vs. matched thyroid normal tissues) and Mann–Whitney U test for independent samples (83 tumors vs. 60 normal thyroid samples). A probability of p < 0.05 was considered as statistically significant. Intraindividual variation was calculated by utilizing the T-to-N ratio that derives from the ratio of corrected tumor (mRNA or protein) levels to corrected normal tissue levels of MDM4 and MDM2.
Results
Analysis of MDM4 and MDM2 levels in papillary thyroid tumors
A group of 57 thyroid tumors samples and 57 matched normal thyroid tissues from the CTRL were analyzed. All tumors were of papillary histotype (PTC). The mean age of the studied population was 49.4 ± 16.81 (SD); the female percentage was 72%. The histopathological features of the 57 tumors are shown in Table 1. Tumor stage was defined according to the sixth edition of the “American Joint Committee on Cancer.”.Table 1Histopathological features of the 57 Papillary Thyroid Tumors (PTC) Yes, n (%)No, n (%)ND, n (%)BRAF mutation35 (61)20 (35)2 (3)Ret/PTC rearrangement5 (9)50 (88)2 (3)TP53 mutation0 (0)57 (100)0Extra-thyroidal invasion15 (26)41 (72)1 (2)Multifocality24 (42)32 (56)1 (2)Nodal metastasis24 (42)33 (58)0Distant metastasis6 (10)35 (62)16 (28)Tumor stage of patients <45 years (n = 20)I15 (75)05 (25)II0 (0)15 (75)5 (25)Tumor stage of patients ≥45 years (n = 37)I8 (22)18 (48)11 (30)II2 (5)24 (65)11 (30)III3 (8)23 (62)11 (30)IV13 (35)13 (35)11 (30)HistologyClassic38 (67)19 (33)0Follicular13 (23)44 (77)0Other4 (7)53 (93)0Dedifferentiated2 (3)55 (97)0ND Not determined
All samples were screened for the presence of TP53 mutations (limitedly to exons 5–8 where 80% of mutations occur), BRAF mutations (by SSCP followed by sequence analysis), and for ret/PTC 1 and ret/PTC 3 rearrangements (by RT-PCR; Table 2). MDM4 and flMDM2 mRNA levels were evaluated by qRT-PCR, using specific primers and probes. MDM4 primers were chosen in a region not present in the variant forms MDM4-S, HDMX-G [19] MDM4-211, XALT1, and XALT2 [20] to amplify specifically the fl product. Similarly, the chosen MDM2 primers do not amplify the MDM2-A, MDM2-B, MDM2-C, MDM2-D, and MDM2-E alternative spliced forms and most of the aberrantly spliced forms [21].
Table 2Tumor sample dataSampleM4M4 T/NMDM4-S/flM4MDM4-211MDM4-211 to flM4M2 T/NM4 to M2TP53 statusBRAF mutRet/PTC rearr.Patient statusaTumor stageMultifocalityN1M1Histologyb19.420.91.81.360.1411.3wt+−13−−−2218.961.35.21.42.5wt−−44+++4329.341.60.49.030.311.53.5wt−−11−−−2414.931.79.71.12wt−+11+−−154.390.30.90.41.1wt−−44−++3611.160.41.30.51.9wt−−44−++3712.600.80.78.940.710.73.9wt−−11++−184.440.40.82.10.7wt+−n.d.n.d.−−n.d.1915.511.40.61.41.6wt+−14++−1108.630.30.60.91.3wt−−n.d.n.d.+−n.d.1115.960.21.20.90.7wt−−24−+−1128.110.51.110.9wt+−31++−1136.250.41.310.161.80.8wt+−n.d.n.d.−+n.d.2146.130.41.50.80.9wt+−n.d.n.d.−−n.d.1158.340.31.60.61.2wt−−11−+−1163.820.21.41.230.320.80.5wt+−31−−−11718.8310.80.38.8wt+−24++−2186.321.20.71.11.2wt+−11−−−1193.610.21.82.60.1wt−−11−−−2205.120.330.90.8wt+−n.d.1+−−1218.40.54.20.61.1wt−+2n.d.+−n.d.1225.10.3n.d.*10.6wt−+13−−−1237.060.90.92.60.7wt+−n.d.n.d.−−n.d.1248.850.60.111.4wt+−11+−−1256.210.60.12.30.6wt−−n.d.n.d.+−n.d.1269.6110.11.21.1wt+−11−−−2277.110.30.60.41.6wt+−n.d.n.d.++n.d.1283.530.2n.d.0.21.4wt+−12−−−2295.760.42.20.71.2wt+−n.d.n.d.+−n.d.13010.7111.91.2wt+−11−−−13111.470.3n.d.0.80.9wt−+31++−13214.421.5n.d.2.41.4wt+−11−−−1333.920.30.20.41.7wt+−34−−+13411.960.710.52.8wt−−11−−−2353.690.11.81.170.320.40.7wt+−14++−2368.030.42.70.71.4wt+−11−−−13721.3320.40.85.6wt−−11++−4385.560.44.71.60.6wt+−34++−2391.120.11.12.40wt−−14−+−24013.981.41.43.50.9wt+−11+−−2415.840.33.610.6wt+−34−++1427.360.31.31.830.250.90.8wt+−11−−−1434.770.31.20.80.8wt−+3n.d.−+n.d.4442.450.12.20.70.4wt+−13−+−1455.120.30.70.80.7wt+−34−++1463.10.32.11.60.2wt+−11−−−14710.271.12.419.60.1wt+−11++−24812.131.12.41.12.2wt+−n.d.n.d.+−n.d.1497.940.81.60.82.2wt−−31++−15014.720.82.80.82.2wt+−34n.d.+−15115.671112.2wt+−12−−−15266.493.60.63.62.2wt+−1n.d.+−n.d.15328.941.41.71.42.2wt+−1n.d.−−n.d.1543.720.40.32.80.5wt−−1n.d.++n.d.4555.30.40.71.80.5wt+−11−−−1562.040.60.92.30.3wtn.d.n.d.1n.d.+−n.d.1576.870.7n.d.1.40.8wtn.d.n.d.1n.d.−−n.d.1n.d. Not determined, N1 nodal metastasis, M1 distant metastasisa1, recovered; 2, recurrence occurred, 3,disease persistence, 4, expiredb1, classic variant; 2, follicular variant; 3, dedifferentiated variant; 4, other
MDM4 and MDM2 mRNA levels in normal tissues followed a normal distribution according to Kurtosis analysis (Fig. 1a,b). On the contrary, MDM4 and MDM2 values in tumor samples were not normally distributed (p < 0.0001; Fig. 1c,d). Comparative analysis by Wilcoxon signed-ranks test for paired samples revealed a highly significant difference in the distribution of MDM4 values between tumor and CTRL groups (Wilcoxon’s W
= 1,385, p < 0.0001; Fig. 1e). Conversely, comparison of MDM2 values did not show significant difference (p
= 0.61; Fig. 1f), in spite of the presence of four upper outliers in the tumor sample set. There was no correlation between MDM4 and MDM2 mRNA tumor levels indicating that the alterations of the two transcripts are independent from one another (data not shown).
Fig. 1a–d Frequency histograms showing the distribution of the mRNA levels of the indicated genes in the indicated sample groups. Relative units of target mRNA are referred to a sample called calibrator, chosen to represent 1× expression of the target gene. Each sample mRNA was normalized relative to the β-actin mRNA. The superimposed line shows the normal distribution curve. For each distribution, the mean (M) ± standard deviation (SD) is shown. e, f Comparison of MDM4 and MDM2 levels, respectively, between control and tumor samples. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Wilcoxon signed-ranks test
To confirm the decrease of MDM4 mRNA levels in PTC in comparison to CTRL, we analyzed these levels in an additional 26 independent PTC and three normal thyroid samples. Comparison of tumor and CTRL groups still resulted in a highly significant correlation (Mann–Whitney for independent samples U
= 634, p < 0.0001) and confirmed previous results of a strong downregulation of MDM4 expression in tumor samples. Indeed, study of prediction by receive operator characteristics curves (ROC curves) indicated that the use of MDM4 mRNA levels as potential diagnostic test has a highly significant ability to discriminate between normal and tumor tissues (area under the ROC 0.84, p < 0.0001 Fig. 2).
Fig. 2Receive operator characteristics curves for MDM4 levels between normal and tumor samples, assessing variable MDM4 levels as diagnostic test. The plot shows sensitivity of the test against specificity. p value was 0.0001. For each decision threshold, the percentage of tumor cases correctly identified as such (true positives) against the percentage of normal cases incorrectly diagnosed as tumor (false positives) is shown
To ascertain whether the decrease of MDM4 mRNA in tumor samples results in decreased protein levels, we analyzed whole-cell extracts from 20 available pairs of tumors and CTRLs (Fig. 3). In 16 out of 20 pairs, the tumor-to-CTRL ratio of densitometric values of the MDM4 protein levels correlated significantly with the observed ratio of the mRNA levels (Kendall tau = 0.7, p
= 0.0002). Interestingly, western blot confirmed the decrease of MDM4 mRNA in tumor relative to normal tissue in seven samples, reinforcing previous observations. Notably, in three samples (44, 27, and 33), we observed an increase of MDM4 proteins in tumor compared to CTRL despite equal or even lower levels of corresponding tumor mRNA relative to CTRL, indicating MDM4 protein stabilization independent of transcription. Western blot analysis revealed also the presence of an additional species of about 58 kDa in some tumor samples (Fig. 3, arrows). This band was recognized also by a mix of three monoclonal antibodies to MDM4 (6B1A, 11F4D, and 12G11G, data not shown), suggesting it may be a MDM4 variant. However, its size does not correspond to any of the MDM4 variant forms described so far.
Fig. 3Western blot of WCE derived from 20 couples of tumor (T) and matching control (N). The values derive from the ratio of densitometric value of MDM4–actin, both at the protein and mRNA levels. Asterisks mark tumor samples expressing MDM4-211 mRNA; section signs mark tumor samples showing increased levels of MDM2 protein with no increased levels of the corresponding mRNA. Arrows mark a lower weight protein of about 58 kDa, recognized by different αMDM4 antibodies
Further, western blot revealed frequent overexpression of the MDM2 protein in tumors that did not show overexpression of the mRNA (Fig. 3, samples marked by section signs).
Analysis of intraindividual variation of MDM4 and MDM2 levels
Previous data were obtained by comparing tumors versus CTRLs. We then analyzed the intraindividual variation of MDM4 and MDM2 mRNA levels for each tumor (T) and its matched CTRL (N) by calculating the relative ratio, T to N. The range of MDM4 T-to-N ratios varied from 0.1 to 3.6 (Fig. 4a and Table 2). The majority of tumor samples showed a decrease of MDM4 levels compared to the corresponding CTRLs (29 out of 57 samples having a ratio of <0.5), confirming the previously observed decrease of tumor MDM4 expression at individual levels too. In other tumor histotypes, amplification of MDM4 gene and overexpression of the mRNA have been reported. In this PTC group, MDM4 overexpression (as defined by a T-to-N ratio of ≥5) was not detectable; in only two cases, the ratio of T to N showed a value of ≥2 (2 and 3.6, respectively). On the contrary, comparison of MDM2 mRNA levels showed tumor values twofold higher than matching CTRL in 11 samples, among which one had a ratio of >19, suggesting an amplification event (Fig. 4b and Table 2). Moreover, only six samples (10%) showed a ratio of <0.5, indicating that downregulation of MDM2 levels in PTC tumor is a rare event in comparison to that of MDM4. Notably, in two of these tumors, western blot analysis did not confirm the mRNA MDM2 decrease (Fig. 3; samples 35, 27).
Fig. 4a, b Frequency histograms showing the distribution of the T-to-N ratios derived from tumor–CTRL levels of the indicated genes. The superimposed line shows the normal distribution curve. For each distribution, the mean ± standard deviation is shown
Correlation of MDM4 and MDM2 with tumor histopathological features
We then investigated whether tumor MDM4 and MDM2 mRNA levels as well as the T-to-N ratio varied according to tumor properties by correlating them with the histopathological features of samples as reported in Table 1. First, we examined their association with BRAF mutations and ret/PTC rearrangements, two hallmarks of thyroid carcinogenesis. BRAF mutation were detected in 35 out of 57 patients, ret/PTC rearrangements in 5 out of 57 (Table 2). All BRAF mutations but one involved a heterozygous T > A transversion at nucleotide 1799, resulting in the prototypic valine-to-glutamic-acid substitution at position 600 (BRAFV600E). In one case, the in-frame insertion at position 1796 of an additional codon, coding for Valine, was detected (BRAFV599Ins). This was shown to be a “gain-of-function” mutation in all super imposable to BRAFV600E [22]. No association was found between MDM4 and MDM2 expression, BRAF mutations, and ret/PTC rearrangements suggesting that these oncogenic hits are independent events. We then analyzed the correlation between MDM4 or MDM2 expression with the other tumor histopathological features. This revealed association of MDM4 with multifocality of tumors: specifically, tumors presenting as multifocal show higher values than nonmultifocal (Spearman rank correlation rs = 0.29, p
= 0.032; Fig. 5a, Table 3). This association was not evident when we correlated multifocality with the MDM4 levels of matching normal tissues (p
= 0.23), indicating that this tumor property is not attributable to a patient predisposition given by individual MDM4 expression levels but rather it is a specific feature of tumor cells.
Fig. 5a Vertical box-whisker plots showing MDM4 levels between nonmultifocal (no) and multifocal (yes) tumors. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman test. b Vertical box-whisker plots showing tumor–CTRL MDM4 mRNA levels in stage 1 compared to other stages of tumors. Each plot shows graphically the central location and scatter–dispersion of the values of each group: the line series shows parametric statistics (mean and confidence interval of mean) while the notched box and whiskers show nonparametric statistics (median, confidence interval of median, and interquartile range). Crosses and circles indicate possible outliers, between 1.5 and 3 interquartile range and over 3 interquartile range, respectively. p value was calculated according to Spearman testTable 3Summary of significant correlations of MDM4 and MDM4 T–N values with histopathological features MultifocalityTumor StagePresenceAbsenceIII–IVn = 24n = 32n = 23n = 18MDM4 T–NMean ± SD0.9 ± 0.760.56 ± 0.430.81 ± 0.540.51 ± 0.43Median ± IQR0.63 ± 0.830.38 ± 0.60.65 ± 0.810.3 ± 0.58MDM4Mean ±SD12.4 ± 12.68.1 ± 6.410.28 ± 5.98.38 ± 5.9Median ± IQR8.74 ± 8.76.19 ± 5.18.85 ± 5.445.7 ± 8.0
We also correlated MDM4 T-to-N ratio with tumor features. We observed an even stronger association between this parameter and multifocality (Spearman correlation coefficient rs = 0.32, p = 0.01, Table 3), confirming previous observations. Further, we found a significant correlation of MDM4 T-to-N ratio with tumor stage (Spearman correlation coefficient, rs = 0.35 for n = 41, p = 0.02; Fig. 5b); in particular, stage I tumors showed the highest ratio, indicating downregulation of MDM4 levels in late-stage tumors in comparison to early-stage ones (Table 3).
On the contrary, MDM2 levels as well as MDM2 T-to-N ratio did not show correlation with any histopathological parameter.
Characterization of MDM4 variants in thyroid tumors
We have analyzed the presence of the MDM4 variant forms, MDM4-S and MDM4-211, in the same sample group. So far, these are the only variant forms of MDM4 detected in human tumors. MDM4-S is a splicing variant present both in normal and tumor tissues; however, in tumor cells, MDM4-S is overexpressed and, as a consequence, the ratio MDM4-S to flMDM4 is >1. At first, we performed comparative analysis of flMDM4 and MDM4-S by qRT-PCR in CTRL samples. In agreement with the literature, we detected both transcripts in all samples with a ratio MDM4-S to fl-MDM4 ranging from 0.09 to 0.79 with only two samples showing values of 1.14 and 1.05 (data not shown). On the basis of these results, we considered overexpression as defined by a ratio of ≥1.5 The same analysis in PTC detected MDM4-S in all but five samples; the resulting MDM4-S-to-flMDM4 ratio was ≥1.5 in 20 out of 57 of tumors (35%; Table 2). The same percentage resulted from the analysis of the additional group of 26 tumors (9 out of 26), in agreement with other reports [11]. Statistical analysis did not reveal any significant association of MDM4-S-to-flMDM4 ratio with the features described in Table 1.
The second variant form we investigated was MDM4-211 (HDMX211), previously isolated from a thyroid tumor cell line [13]. MDM4-211 mRNA was present in 7 out of 57 original tumor samples and in 8 out of 26 additional samples analyzed with an overall frequency of 18% (Table 2 and data not shown). Its mRNA was not detected in any of the CRTL samples, thus confirming its specific presence in tumor tissue. In agreement with our previous reports [13], western blot analysis of available tumor and CTRL samples confirmed enhanced levels of the oncoprotein MDM2 in tumors expressing MDM4-211 (Fig. 3, samples marked by *). In our tumor set, the presence of MDM4-211 did not correlate with any of the properties described in Table 1 nor with the expression of the form MDM4-S, indicating that the presence of these two variants is an independent event.
Some authors have hypothesized that the presence of MDM4 variants may correlate with lower level of flMDM4 [11]. We therefore assessed the presence and expression of MDM4-S and MDM4-211 in correlation with flMDM4 and with the MDM4 T-to-N ratio. We observed no significant correlation but rather a slight association between low levels of both MDM4-S and flMDM4 (p = 0.056).
Discussion
In this study, we have analyzed the expression of MDM4 and MDM2 mRNAs in a group of 57 papillary thyroid tumors in comparison to matching normal tissues. We observed that MDM4 levels are significantly lower in tumor samples than in control. Specifically, tumor samples expressing levels of MDM4 lower than matching control are very frequent (51%) and this decrease in expression appears to correlate significantly with higher tumor stages (stage I in comparison to the other), indicating that it occurs with tumor progression. These data led us to hypothesize that a robust presence of MDM4 might exert an antagonistic role in PTC evolution. To date, the majority of the literature has reported amplification of MDM4 in the presence of wt TP53 in different tumor histotypes [3], partly in contrast with our data. However, a different status of MDM4 in some cancer has also been reported. In a gene profiling analysis, MDM4 downregulation was observed in prostate tumors with poor prognosis in comparison to those with good prognosis in agreement with the hypothesis that MDM4 may be detrimental to tumor progression [23]. Moreover, two studies reported low levels of MDM4 protein in chronic myeloid leukemia tumor samples and in tumor cell lines [24, 25]. It would be interesting to know whether those cell lines have features resembling those of advanced-stage tumors. Some authors have suggested that low MDM4 expression levels may be correlated to the presence of a mutated TP53 [11, 25]. However, lack of detection of any of the most frequent mutation of TP53 gene in this PTC group does not support this hypothesis, at least in this type of cancer. Similarly, in the prestate tumor study TP53 was found to be wild type by sequencing (A. Farsetti, personal communication).
These data counteract the holistic view of MDM4 as a p53 inactivation tool and suggest that its function may be more complex than previously considered, at least in some tissues. Thus far, molecular studies indicating MDM4 functions different from negative regulation of 53 have provided evidence that MDM4 may display antiproliferative or proapoptotic activities, under stress conditions [6, 7]. Further, a recent work has shown for MDM4 a role in suppressing tumorigenesis through the control of bipolar mitosis [26]. Whether these mechanisms occur in PTC remain to be elucidated.
In the analysis of tumor MDM4 expression levels, we found that high levels of MDM4 are associated with multifocal tumors. Different hypothesis have been postulated to explain the origin of these tumors. Certainly, multifocality is related to the high proliferative potential of thyrocites. MDM4 has been reported as inhibitor of p53-mediated growth arrest [1]; thus, it might be hypothesized that the presence of MDM4 may predispose to multifocality and tumor development, while a decrease in its expression may confer an advantage to tumor progression. Interestingly, one study has documented a statistically significant association of MDM4 amplification with low-grade astrocytomas, thus supporting the hypothesis that high levels of MDM4 may represent an early advantageous event for tumor development [27]. Further, the association between MDM4 levels and tumor multifocality is of particular clinical relevance. Indeed, the possibility of early diagnosis of tumor multifocality might allow discrimination between partial or total thyroid gland ablation. The extension of this type of analysis to a broader set of samples, including benign lesions, should help to confirm these findings and to investigate potential applications of this molecule in clinical practice.
In addition, we have observed with high frequency the aberrant presence of MDM4 alternatively spliced forms, whose oncogenic activity has been assessed so far only in vitro. In our analysis, the aberrant presence of these variants did not correlate with any of the histopathological features. In a study with primary sarcomas, the MDM4-S-to-flMDM4 ratio correlated with enhanced cell proliferative potential and decreased patient survival [11]. The high incidence of patient survival in PTC (only three deaths in our group, Table 2) does not allow to confirm this association. For MDM4-211, this is the first frequency report in a large group of tumor samples and indicates that its presence is appreciable in PTC (18%). Interestingly, these MDM4 variants forms show in vitro an even stronger inhibitory activity toward p53 than the full-length protein. Their presence may concur to modify the activity of flMDM4, making more complex the assessment of its function in tumor cells. It has to be mentioned that in many PTC samples we observed the presence of a MDM4 minor form whose nature is unclear, supporting the hypothesis that MDM4 variants may concur to MDM4 oncogenic functions.
Conversely, MDM2 mRNA levels were increased in majority of tumor samples. In particular, we observed with high frequency enhanced protein levels in tumor samples that do not show enhanced mRNA levels. This is in accord with previous reports and confirms that in thyroid tumors MDM2 overexpression takes place frequently by mechanisms other than enhanced transcription [28]. At least in some of these samples, the simultaneous presence of MDM4-211 variant may cause such upregulation. These data further confirm the general view of the antagonistic role of MDM2 toward the oncosuppressor p53. Finally, in this study, we did not find any correlation between MDM2 and MDM4 mRNA levels, suggesting that the alterations we observed occur independently from one another.
Overall, our results reveal that MDM4 alterations, particularly the presence of its variant forms with p53-inhibitory properties, are present at high frequency in PTC confirming the general model of different ways of p53 inactivation in human tumors. However, our data represent the first statistical report of a strongly significant downregulation of flMDM4 expression in cancer and particularly in advanced-stage tumors, suggesting that this protein may play additional roles besides p53 inactivation at least in PTC. In view of the suggested strategies for abrogation of MDM4 in human tumors [1, 3], our findings counsel acquisition of more information on the molecular functions of this protein in different human tumors in order to carefully evaluate the application of such therapies.


BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.
Introduction
Cartilage–bone transition by endochondral ossification is a highly complex process which determines not only longitudinal growth of long bones, ribs and vertebrae, but also plays a critical role in bone fracture callus healing, osteophyte formation and cartilage tissue engineering. Decisive steps in this process are proliferation, maturation and hypertrophy of chondrocytes in the growth plate which play a key role in mineralization, apoptosis and induction of resorption of hypertrophic cartilage by invading bone marrow. These events are regulated by synergistic action of several signaling pathways and transcriptions factors controlled by a multitude of growth factors and hormones.
A major signaling system regulating chondrocyte proliferation and maturation is controlled by PTHrP, which is induced by Indian hedgehog, stimulates chondrocyte proliferation and delays maturation through the PTHrP receptor PTHR1 [for reviews see (Kronenberg et al. 1998; Vortkamp 2000)]. Also FGF factors stimulate proliferation and block maturation through FGF receptor 3, but their action in the growth plate is more complex with respect to the differential distribution of different FGFs and FGF receptors in the various zones of the growth plate (Ornitz and Marie 2002).
The role of BMPs during chondrocyte maturation and hypertrophy is somewhat controversially discussed: several studies show that BMP factors such as BMP-6 and BMP-7 stimulate hypertrophic differentiation of chondrocytes and promote collagen X expression, thus preparing growth plate cartilage for replacement by endochondral bone (Enomoto-Iwamoto et al. 1998; Volk et al. 1998; Grimsrud et al. 2001). On the other hand, it was reported that BMP-2 and BMP-4 overexpression in developing chick limbs caused delayed hypertrophy of chondrocytes (Duprez et al. 1996).
The fourth family regulating chondrocyte maturation are Wnt factors and the Wnt/β-catenin signaling system (Dong et al. 2005), also acting in diverse directions. For example, Wnt 7a inhibits chondrogenesis and Wnt 5a impairs chondrocyte hypertrophy and type X collagen (Col10a1) expression in vitro (Daumer et al. 2004), but activation of the canonical β-catenin pathway by Wnt 8c and Wnt 9a induces chondrocytes maturation, hypertrophy and calcification, associated with upregulation of Col10a1 expression (Enomoto-Iwamoto et al. 2002; Dong et al. 2005, 2006; Hu et al. 2005).
Recently increasing evidence points to complex crosstalks between these signaling pathways. For example, there is experimental evidence for a coordination between Indian hedgehog and BMP signaling in chondrocyte proliferation (Minina et al. 2002). Targets of the signaling pathways in the growth plate are transcription factors such as Sox9 responsible for chondrogenic differentiation (Ng et al. 1997; Lefebvre and de Crombrugghe 1998) and cbfa1/runx2 inducing osteogenic differentiation (Karsenty et al. 1999; Ducy 2000) as well as chondrocyte maturation and Col10a1 expression (Kim et al. 1999; Enomoto et al. 2000; Zheng et al. 2003).
There is ample evidence in the literature that factors regulating chondrocyte maturation and hypertrophy may be also directly involved in the regulation of Col10a1 gene expression, e.g. PTHrP (Iwamoto et al. 1994; Ionescu et al. 2001; Riemer et al. 2002; Gebhard et al. 2004), Runx2 (Enomoto-Iwamoto et al. 2001; Leboy et al. 2001; Zheng et al. 2003), BMP2 (Volk et al. 1998; Grimsrud et al. 2001), or c-fos (Thomas et al. 2000; Riemer et al. 2002). Thus, in order to elucidate the exact role of the various signaling systems and involved factors regulating endochondral ossification, it would be useful to generate a reporter gene mouse in which the expression of LacZ is under the control of the complete Col10a1 gene regulatory sequences.
Previously we have reported on the generation of transgenic mouse lines in which LacZ expression is under the control of the 4.6 kb promoter of Col10a1 which includes a strong enhancer element (Gebhard et al. 2004) and runx2 binding sites (Zheng et al. 2003). We have shown that this promoter is sufficient to drive specific expression of LacZ reporter genes in hypertrophic chondrocytes of growth plate cartilage of long bones, ribs, vertebrae and sternal cartilage (Gebhard et al. 2004). Reporter gene expression was restricted to hypertrophic cartilage, but not all hypertrophic chondrocytes in the growth plate of 18.5-day embryos were found to be lacZ positive, and only weak lacZ expression was observed at early embryonic stages before day E17. Furthermore, not all transgenic founders containing the LacZ transgene showed lacZ activity in the growth plates, suggesting a high level of sensitivity to interfering activities of the genomic context of insertion or additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences.
Here we report on the construction of a BAC reporter mouse expressing lacZ under the control of the Col10a1 gene. A LacZ-Neo cassette was inserted into the second exon of Col10a1 within the context of a 215 kb BAC using a phage-based homologous recombination system in E. coli (Yu et al. 2000; Lee et al. 2001). Transgenic mouse lines established with this modified BAC show specific LacZlacZ expression at high levels in hypertrophic zones of long bones, ribs, vertebrae, mandibles and sterna of transgenic mouse lines. No significant unspecific expression was detected in other chondrogenic or non-chondrogenic tissues except some transient, probably unspecific X-gal reaction in the prenatal epidermis and hair papillae.
The robust and specific expression of Col10a1-based BAC recombineering vectors in transgenic mice opens new and unique possibilities to study the role of growth factors and transcription factors in chondrocyte hypertrophy and endochondral ossification, and to define further cis-acting regions in the Col10a1 gene.
Materials and methods
BAC clones
Murine Col10a1 containing BAC clones were selected from the UCSC Bioinformatic databases (http://www.genome.ucsc.edu/) (Kent et al. 2002); clone RP23-192A7 contained 211 kb of genomic DNA flanking the Col10a1 gene derived from the L129 mouse strain. RP23-192A7 (cloned in the pBACe3.6 vector) was obtained from the BACPAC Resources Center at Children’s Hospital Oakland Research Institute, (CHORI). The presence of the full-length Col10a1 gene was verified by PCR amplification using both primers for detecting the enhancer at −4.6 kb (Enmcol10a1-3′: ATT CTC GAG CTT TGG GAA GCA TGG TG ; Enmcol10a1-5′: CAA GAA ATC TTT GGG AAA TGA ATG AAT G ) (Gebhard et al. 2004) , and for a region 2036 bp downstream of the poly A site of the Col10a1 gene (mcol10a1-psg-5′: TCT ACC AGG AGG CCT CTC TTC AAT GTT AC; mcol10a1-psg-3′:
GGG AAT TCT TAC CTT AAA GTA GAT ACA TG (amplicon size 222 bp).
Construction of targeting vector placH+Col10a1-lacZ-frt-neo-frt
The 5′ recombination site was generated by PCR amplification of a 129 bp fragment mapping to the 113 bp 3′-end of intron 1 and 16 bp of the 5′-UTR encoded in exon 2 by using BAC RP23-192A7 as template (see Fig. 1). Terminal SalI and NcoI restriction sites were added by using primers Col10a1-5′SalI (5′-ACG CGT CGA CGA TAT CTC AGT CAT TTA AAA AAC CAT GA) and Col10a1-5′NcoI (5′-CAT GCC ATG GAT TTT CAG ATA GAT TCT GAA AAG CAG A. The product was digested with SalI and NcoI and cloned into the multiple cloning site 2 of plasmid placH (Zhou et al. 1995) kindly provided by Dr. V. Lefebvre, Cleveland) containing the lacZ gene and the 3′-end of the murine protamine 1 gene with an intron and poly A signal resulting in clone placH+5′COL10a1. Use of the NcoI site linked the lacZ reading frame to the start ATG of Col10a1.
Fig. 1Generation of the BAC-Col10a1-lacZ-neo DNA for the transgenic expression of the lacZ reporter gene in hypertrophic cartilage. a Genomic structure of the murine Col10a1 gene, with exons (gray boxes) and introns. The sequences used for generation of the 5′ and 3′ homologous regions in exon 2 and intron 1 are shown in black. b The targeting vector placH- Col10a1-lacZ-frt-neo-frt contains Col10a1 derived 5′ and 3′ homology domains flanking a lacZ-frt-neo-frt cassette for homologous recombination in E. coli. The lacZ coding sequence was fused with the start ATG of the Col10a1 reading frame; shown is only the insert of the targeting vector, contained in the placH vector. c Partial map of the BAC-clone Col10a1-lacZ-frt-neo-frt clone after homologous recombination with the targeting vector placH-Col10a1-lacZ-frt-neo-frt
The 3′ recombination site was generated by overlapping primers corresponding to a 57 bp sequence of exon 2. Additional HindIII sites were introduced at both ends as well as a XhoI site at the 3′-end. The frt-neo-frt cassette of the pICGN21 vector (Yu et al. 2000; Lee et al. 2001); kindly provided by Dr. Copeland) harboring the neomycin phosphotransferase gene under the SV40 promoter, an HSV thymidine kinase and a poly A signal was amplified by PCR using primer FRTNeo5´HindIII (5′-AGT CAA GCT TTA ACT GAT CGC GGC CAG CTT GAA GTT) (pIGCN21 sequence pos. 4664–4689, marked in bold) and the primer Col10a1-3′HindIII (5′-TAA AGC TTC TCG AGT TGT GTC TTG GGG CTA GCAAG TGG GCC CTT TAT GCC TGT GGG CGT TTG GTA CCG TTC TAT TCC AGA AGT AGT GAG GAG GCT TT) and plasmid pICGN21 as template (pICGN2 sequences pos. 6103 to 6123 marked in italics) and Col10a1 from exon 2 (82 to 138, underlined). The product was digested with HindIII and inserted into the multiple cloning site behind the mP1 gene of placH+5′Col10a1. The resulting plasmid placH+Col10a1-lacZ-frt-neo-frt was verified to be correct by restriction digestion and sequencing for correct insertion and composition.
Homologous recombination and detection of BAC transgene
BAC RP23-192A7 was electroporated into the E. coli strain EL250 (DH10B[λcl857(cro-bioA)<>araC-PBADflpe]) kindly provided by Dr. Neal G. Copeland, NCI, Frederick, MD, USA, containing a defective λ prophage which supplies essential functions (exo, bet, gam) to protect and enhance recombination of linear DNA (Lee et al. 2001).
The 5.1 kb recombination cassette was excised from plasmid placH+COL10a1-lacZ-frt-neo-frt by XhoI; complete linearization with XhoI was important to avoid amplification of the circular targeting vector in E. coli.
For homologous recombination 300 ng linear fragment was transformed by electroporation according to Dower (Dower et al. 1988) in 0.1 ml cuvettes containing 50 μl of ice-cold competent EL250 containing BAC RP23-192A7, using a Bio-Rad gene pulser set at 1,750 kV, 23 mF with a pulse controller set at 200 Ω. Electrocompetent bacteria were prepared as described in (Lee et al. 2001). A 15 min heat shock at 42°C was used for induction of the homologous recombination machinery according to (Yu et al. 2000; Lee et al. 2001). Transformed cells were incubated for 1.5 h at 32°C in 1 ml of LB and selected for chloramphenicol- and kanamycin-resistance. Resulting clones (RP23-192A7 + lacZ-frt-neo-frt) were screened for homologous recombination by PCR detecting the 5′ recombination event (amplicon 437 bp) using primers P1 (5′-TTT AGA GCA TTA TTT CAA GGC AGT TTC CA) and P2 (5′-CGG CAC CGC TTC TGG TGC CGG AAA CCA GGC), and the 3′ recombination event (product 378 bp) using primers P3 (5′-ACA GAA TAA AAC GCA CGG GTG TTG GGC GT) and P4 (5′-ATC ATT CCG CTG TAC TAG CTC AAG CCA ATC). Direct sequencing of BAC DNA was performed with primers P1 and P4.
BAC DNA purification and generation of transgenic mice
BAC-Col10a1 - lacZ-neo DNA from clone#11 was extracted from multiple E. coli EL250-BAC#11 minipreps by alkaline lysis (Sambrook et al. 2001), purified by potassium acetate precipitation, washed with ethanol and dissolved in Tris-EDTA (TE) buffer. For size and quality control, aliquots were subjected to pulsed field gel electrophoresis (PFGE) (Fig. 2). For purification, 50 μg BAC DNA of clone#11(BAC-Col10a1-LacZ-neo) were dissolved in TE buffer and concentrated to 500 μl by vacuum centrifugation. The DNA was linearized overnight with PISceI enzyme (NEBiolabs) cleaving the unique site in the BACe3.6 vector part. For chromatographic purification a 5 ml plastic pipette was packed with Sepharose CL-4B (Pharmacia) equilibrated in microinjection buffer (5 mM Tris/HCl, pH 7.4, 0.1 mM EDTA, 5 mM NaCl) (Zeilhofer et al. 2005), the linearized BAC#11-DNA was mixed with bromophenol blue and chromatographed. Half milliliter fractions were collected until the dye reached the column bottom, and BAC DNA concentration in each fraction was analyzed by PFGE using serial dilutions of BAC RP23-192A7 DNA as internal standard. The BAC DNA concentration of the main fraction was adjusted to 1.5 ng/μl, and NaCl concentration was increased to 100 mM to stabilize BAC DNA. Fractions were stored at 4°C until microinjection. Injection into fertilized oocytes and generation of transgenic mice was performed by standard techniques using oocytes from FVB mice and FVB/ FVBxC57BL6 F1 hybrids as described (Zeilhofer et al. 2005).
Fig. 2Characterization of one of the BAC-Col10a1-LacZ-frt-neo-frt clones (clone#11). Digestion with PISceI (lane 3), NotI (lane 4), PmeI (lane 5) and NruI (lane 6) resulted in fragments of the expected sizes. Sizes are compared with 1 kb marker (lane 1), 1 kb extension (lane 2) and PFG marker (lane 7), and the lengths are indicated. For microinjection, BAC #11 DNA was linearized with PISceI, purified by gel chromatography on Sepharose CL4B equilibrated in microinjection buffer and analyzed and quantitated by pulsed field gel electrophoresis
Genotyping of transgenic mouse lines
Transgenic founder animals were selected by PCR of tail biopsies using primers pairs P1/P2 and P3/P4 (see above) detecting recombination events. Each sample was standardized by PCR using P1/P4 primer pair which generates a 538 bp fragment from the wt Col10a1 allele. Founders derived from injections of F1 hybrids were crossed with C57BL6, whereas founders derived from FVB oocytes were crossed with FVB mice. For genotyping of newborn mice and embryos DNA was extracted from skin or placenta, and transgene DNA was identified by PCR as above.
Histological analysis and in situ hybridization
Newborns mice were anesthetized under CO2, dissected and subjected to X-gal staining, followed by either clearing with KOH fixation, embedding into paraffin or by cryotome sectioning as described (Zhou et al. 1995). Embryos at 12.5 to 16.5 day were left undissected and stained with X-gal. Paraffin and frozen sections were counterstained with eosin.
In situ hybridization for collagen X was performed as described previously (Aigner et al. 1992), but using a digoxigenin-labeled mouse Col10a1-specific anti-sense probe derived from the 3′-end of the Col10a1 gene instead of 32P-labeled dCTP. Non-radioactive in situ hybridization was done as described elsewhere (Schmidl et al. 2006), using alkaline phosphatase labeled anti-digoxigenin antibodies and BM purple (Roche) as color substrate according to the manufacturers protocol.
Determination of transgene copy number by Southern hybridization
For quantitative analysis of transgene copy number, genomic DNA was isolated from skin of newborns or from placenta of embryos and cleaved with BamHI. For standardization 5 μg wild-type DNA were mixed with 0.37 ng up to 18.38 ng RP23-192A7 + lacZ-frt-neo-frt DNA corresponding to 1 up to 50 copies of recombined BAC per lane. Samples for standardization and 5 μg of DNA from transgenic founders were cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized to with a 32P-labeled probe derived for from the pBACe3.6 vector, prepared by PCR using primers BAC11-5 (5′-TTTAAACGTGGCCAATATGGA) and BAC11-6 (5′-CGCGGATCCTCTCCCTAT). An 8 kb band was detected in transgenic samples and the intensity was measured in a FLA-3000 phosphoimager (Fujifilm) using the AIDA program (Advanced Image Data Analyzer, Raytest ).
Results
Construction of a placH-Col10a1-lacZ-frt-neo-frt targeting vector and homologous recombination
The BAC clone RP23-192A7 selected from the CHORI BAC library RPCI23 contains the mouse Col10a1 gene (7.2 kb) with 171 kb of upstream and 33 kb of downstream genomic regions. For insertion of the lacZ reporter gene into the second exon of the Col10a1 gene by homologous recombination, a targeting vector was constructed containing the lacZ gene linked to a neomycin resistance cassette flanked by two frt sites. The targeting vector was flanked by a 129 bp 5′-terminal homology arm overlapping parts of the 3′-end of intron 1 and 14 bp of exon 2 including the start ATG for fusion with NcoI. The 3′ recombination site mapped to a 57 bp sequence in the 3′ part of exon 2 (Fig. 1b). The vector construct was verified by restriction mapping, Southern blotting and sequencing. Initial attempts using shorter homology regions resulted in mostly incorrect recombination events.
For homologous recombination, the BAC clone RP23-192A7 was electroporated into the E. coli strain EL250, resulting in EL250/RP23-192A7. The Col10a1-lacZ-frt-neo-frt insert was excised from the targeting vector with XhoI (Fig. 1) and electroporated into electrocompetent EL250/RP23-192A7. Chloramphenicol- and kanamycin-resistant BAC-positive clones were selected and tested by PCR for homologous recombination by using two primer pairs P1/P2 and P3/P4 (see Fig. 1 and Materials and Methods) specific for the 5′- and 3′- recombination sites, respectively. To verify the integrity of the Col10a1 gene in its genomic context after homologous recombination, the presence of the enhancer at 4.6 kb upstream the transcription site and the 5′-end of a pseudogene downstream of the 3′ UTR sequences was verified by PCR with specific primers (see Fig. 1 and Materials and Methods) and sequencing of the BAC DNA. Furthermore, intact BAC ends were confirmed by PCR, using primers derived from the pBACe3.6 vector and the genomic insert.
To minimize BAC degradation, DNA was prepared from BAC-Col10a1-LacZ-Neo clone #11 by alkaline lysis and ethanol precipitation, avoiding absorption to minicolumns and shear stress. Control of the BAC DNA by PFGE confirmed the expected size of 220 kb (Fig. 2).
Generation of transgenic mice
DNA from BAC-Col10a1-lacZ-neo (clone #11) was linearized with PISceI at the unique restriction site in the pBACe3.6 vector sequence, purified by molecular sieve chromatography, and injected into the pronuclei of fertilized oocytes of FVB mice and FVB/C59Bl F1 hybrids. Both strains were by far superior in litter size and successful raising offspring as compared to C57/Bl6 mice. Out of 70 newborn pups, 13 were found harboring the lacZ gene after PCR analysis of genomic DNA, using primer pairs P1/P2 and P3/P4 (see Fig. 1). Southern blot analysis of genomic DNA (Fig. 3a) as well as Real- time PCR analysis of genomic DNA using Col10a1 intron specific primers showed that the founders contained between one and seven transgene copies (Table 1)
Fig. 3a Analysis of BAC transgene copy numbers in 13 transgenic founders by Southern hybridization. Genomic DNA was isolated from skin of newborns, cleaved with BamHI, separated on 1% agarose gels and blotted onto nylon membranes. Membranes were hybridized with a 586 bp 32P-labeled probe derived from the pBACe3.6 vector which recognizes an 8 kb band in transgenic samples. Band intensities were quantitated in a phosphoimager. Copy numbers of transgene BAC DNA (Table 1) were calculated on the basis of a standard curve established with serial dilutions of BAC DNA starting from 50 copies down to 1 copy of BAC RP23-.192A7, diluted in 5 µg of wt DNA. b Comparison of LacZ activity in the scapulae of three different BAC transgenic lines harboring different BAC copy numbers shows some correlation between staining intensity and BAC copy number. All specimen were from new born animals and stained under identical conditions with X-gal for 18 hTable 1BAC transgene copy number in 13 founders, determined by Southern hybridization (see Materials and methods)Founder #Strain sexβ-gal activityBAC copy no.800FVB, m+5986FVB, f+31501FVB, f+11504FVB, m+31508F1, fND21510F1, mND11515F1, fND41516F1, f+61520F1, m+71524F1, fND21527F1, mND11533FVB, mND11534FVB, m+4ND not determined
Seven of the 13 independent founder lines were analyzed so far for LacZ activity by X-gal staining of newborns or embryos (all F1 generation). All offspring analyzed revealed strong LacZ activity after X-gal staining which was restricted to the hypertrophic zones in all growth plates of long bones, ribs, vertebrae, sternebrae and to hypertrophic cartilage in the skull in E14.5- and E16.5-day embryos and newborns (see Figs. 4, 5, 6). The X-gal staining pattern was identical in all seven lines investigated, while staining intensity varied in relation to the BAC transgene copy number (Fig. 3b). Under identical staining conditions, X-gal staining was strongest in founder #1520 (seven transgene copies), followed by #800 (five copies) and #986 (three copies) (Fig. 3b).
Fig. 4Detection of lacZ expression in BAC-Col10a1-lacZ-Neo transgenic embryos by X-gal staining at E14.5 (a), E16.5 (b) and P1 (e) of the F1 generation of founder lines #1534 (a), #1504 (b), and #1520 (c). X-gal staining reveals strongest expression in chondrocytes in the hypertrophic zones of the scapula (s), humerus (h), femur (f), clavicula (cl) and all other long bones, furthermore in the hypertrophic zone of the ribs and in cervical vertebrae (c1, c2), (p pelvic bone). c, d In E16.5 embryos some X-gal staining was also seen in the epidermis of the tail (barrow) and in hair papillae of the forehead and snout, and at nostrils (n). This expression is restricted to some basal keratinocytes in the epidermis, here shown in a section of the snout (d). It disappears after birth and may be a result of unspecific β-gal activity. No LacZ activity was detected in non-hypertrophic chondrocytes or in non-chondrogenic tissues.Fig. 5X-gal staining of transgenic newborn mice of BAC-Col10a1-lacZ-neo (a, b founder line #800, F1; c–i founder line #1520, F1). All hypertrophic zones of the growth plates of long bones, ribs, and vertebrae exhibit strong LacZ activity. a Humerus, distal; b ulna, proximal; c forearm, d hand; e skull: C1 cervical vertebrae 1 (atlas); eb ecto-occipital bone; h hyoid bone; m mandibular condyle; MC Meckels cartilage; mh head of malleus; r head of ramus. f Thoracal vertebrae (left four) and cervical vertebrae (right three), view from ventral; g ribs; h spine, view form dorsal, i sternumFig. 6Paraffin sections of transgenic newborns (a–e) (founder line #1520, F1) and E16.5 day (g) (founder line #1534) embryos showing strong and specific LacZ activity in all hypertrophic chondrocytes, e.g. in the growth plate of the humerus (a) (distal), elbow joint of the radius (b), sternebrae (c), atlas (d), ribs (e) and spine (f). Comparison of the lacZ staining pattern in the elbow joint (h radius) with that observed after in situ hybridization with a Col10a1 probe (g) in a parallel section illustrates that LacZ is expressed in the BAC transgenic mice only in collagen X expressing hypertrophic chondrocytes
Specific expression of the BAC-Col10a1-LacZ-neo transgene in hypertrophic cartilage
Various stages of embryonic development were analyzed for expression of the BAC-Col10a1-lacZ-neo transgene by X-gal staining. In E14.5-day embryos, strong LacZ activity was seen in all ribs, the hypertrophic zones of scapula, humerus, radius and ulna in the forearm and femur, tibia and fibula of the hindleg, as well as in mandibles and cervical vertebrae (Fig. 4a). At day 16.5, the extent of X-gal staining expanded in the diaphyseal region of scapula, humerus and in the ribs, corresponding to the expansion of the hypertrophic zones at this embryonic stage. (Fig. 4b, e) Sectioning of X-gal-stained embryos revealed that the lacZ transgene expression in the skeleton occurred exclusively in the hypertrophic zone of growth plates and ribs (see Figs. 5, 6), correlating with collagen X expression as seen by in situ hybridization (Fig. 6g, h).
At embryonic stages E12.5 to 16.5 X-gal staining also occurred in some regions of the epidermis (Fig. 4b, arrow, Fig. 4c, d), mainly in the papillae of the forehead in the snout (Fig. 4c), and in the skin of the tail, hindlegs, forelegs, and shoulder It was restricted to the basal layer of keratinocytes (Fig. 4d) and seen at E12.5 day before the first appearance of hypertrophic chondrocytes in cartilage, but was not detected after birth. It seemed unspecific as no α1(X) mRNA signals were seen in the epidermis or hair papillae at any stage of development by in situ hybridization (N. Adam and K. von der Mark, unpublished data).
Transgenic newborn F1 offspring of all seven founders analyzed so far revealed an even and strong LacZ activity in all growth plates of long bones (Figs. 4e, 5a, b), ribs (Figs. 4e, 5g), vertebrae (Figs. 4e, 5f, h), and sternebrae (Figs. 4e, 5i). In the skull, LacZ activity was seen in mandibular condyles, in hyoid cartilage, heads of malleus and ramus, ectooccipital and basisphenoid bone anlagen, cervical vertebrae and other ossifying cartilage anlagen (Fig. 5e). The high intensity of X-gal staining allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage, for example the inner and outer aspects of the thoracal and cervical vertebrae (Fig. 5f, h).
Analysis of the X-gal staining pattern in paraffin and frozen sections of transgenic embryos and newborn mice confirmed that the LacZ activity was restricted to hypertrophic chondrocytes (Fig. 6), shown here for the humerus (a), radius (b), sternum (c), atlas (d), and ribs (e, f). The X-gal staining pattern correlated well with the expression pattern of α1(X) collagen mRNA seen by in situ hybridization analysis of a similar section (Fig. 6g, h). Neither non-hypertrophic chondrocytes nor any non-chondrocytic cell types revealed detectable levels of LacZ-activity in newborn transgenic animals. After prolonged staining, a non-specific reaction appeared in osteoclasts of long bones in transgenic and wildtype animals owing to endogenous galactosidase activity. Regardless of the transgene copy number, the LacZ activity in the offspring of all founders was seen persistently in all hypertrophic chondrocytes of the developing skeleton.
Discussion
In a previous report on the generation of Col10a1-specific reporter gene mouse lines the expression of lacZ under the control of a 4.6 kb mouse Col10a1 promoter region was restricted to hypertrophic cartilage, indicating that the -4.6 kb mouse promoter including a 500 bp enhancer element located at the 5′-end of this region is sufficient for tissue specific expression (Gebhard et al. 2004). Also in another transgenic mouse expressing LacZ under a −4.0 kb proximal Col10a1 promoter lacking the enhancer, lacZ expression was restricted to hypertrophic cartilage, although mostly to the lower hypertrophic zone (Zheng et al. 2003). The Col10a1 enhancer has been originally described in the human COL10A1 gene; it was located between −1.9 and −2.4 kb by in vitro transfection studies in primary hypertrophic chondrocytes with various reporter gene constructs (Thomas et al. 1995; Beier et al. 1997; Chambers et al. 2002; Riemer et al. 2002). This enhancer is active in hypertrophic, but not in resting zone chondrocytes. Further upstream, a silencer element between −2.4 and −2.8 kb suppressed transcription in non-chondrogenic cells (Beier et al. 1997). The enhancer element is highly conserved in mammals with 78–85% sequence identity between human, bovine and murine Col10a1 genes (Gebhard et al. 2004) and contains a conserved AP-1 site which is essential for high transcription of enhancer- reporter genes in hypertrophic chondrocytes (Gebhard et al. 2004). In the murine gene, the enhancer is located between −4.1 and −4.6 kb due to insertion of two virus-related sequences in the promoter (Gebhard et al. 2004). Further cis-regulatory elements in the 4.6 kb promoter include an AP-1 site in the proximal promoter (Harada et al. 1996) and several runx2 bindings sites (OSE elements), (Zheng et al. 2003).
Yet, although lacZ transgene expression in the 4.6 kb Col10a1–LacZ reporter mice was specific and restricted to hypertrophic chondrocytes, not all hypertrophic chondrocytes in the growth plates of 18.5-day embryos were found to be LacZ positive, and only weak LacZ expression was observed at early embryonic stages before day E17 (Gebhard et al. 2004). Furthermore, not all transgenic founders containing the lacZ transgene were stained with X-gal in the growth plates, suggesting positional effects of additional regulatory elements in the Col10a1 gene further upstream of the enhancer or downstream of the coding sequences. It may also indicate a high level of sensitivity to interfering activities of the genomic context of insertion. It is possible that the −4.6 kb promoter including the first intron contains essential silencing elements required for suppressing Col10a1 expression in non-hypertrophic cartilage and in non-chondrogenic tissues, but possibly lacks additional regulatory elements beyond the enhancer region.
In contrast, in the Col10a1-BAC transgenic lines presented here, all hypertrophic chondrocytes showed strong and even LacZ expression in all zones of endochondral ossification in embryonic and postnatal transgenic animals. The LacZ reaction of hypertrophic cartilage became visible already after 3–4 h of X-gal staining, while under the control of the 4.6 kb Col10a1 promoter significant LacZ activity in transgenic mice did not become visible before 18 h staining time (Gebhard et al. 2004).
Recently, evidence is accumulating that in many genes further tissue-specific enhancers are located in large distances from the start site of transcription. For example, regulatory sequences were identified 20–30 kb upstream of the transcription start site of the Myo D gene (Goldhamer et al. 1992) or the Ubx gene in Drosophila (Irvine et al. 1991), up to 270 kb downstream in the mouse BMP5 gene (DiLeone et al. 2000) or between 290K kb upstream and 450 kb downstream of the Sox9 gene (Bagheri-Fam et al. 2006). Thus we cannot exclude that additional tissue-specific regulatory elements may be present in the Col10a1 gene, either upstream in the region between −170 kb and the enhancer at −4.6 kb, or downstream of the 3′-end of the Col10a1 gene. The BAC transgenic reporter lines established here would facilitate the identification of such elements by generating further mutants using the homologous recombination technique (DiLeone et al. 2000).
Reasons for the high reporter gene expression levels in the BAC-Col10a1-lacZ-neo reporter lines in comparison to the 4.6 kb Col10a1-lacZ mice are for one part the high copy number of BAC transgenes, and for the other part the favorable und undisturbed conditions for transgene expression in the BAC environment. The X-gal staining intensity in founder line #1520 harboring seven BAC transgene copies was clearly higher than that of founder line #986 harboring only three BAC copies. But in addition, the use of targeted BAC recombination permits the expression of genes in their genomic context, containing most relevant regulatory regions in the correct orientation and distance, in contrast to conventional transgenic mice generated by injection of short reporter genes. This strategy is less sensitive to unpredictable influences by regions surrounding the random integration site (Mortlock et al. 2003). The expression pattern of LacZ, however, was very similar in all seven BAC lines analyzed so far, apparently independent of the integration sites and the copy number of the BAC transgene.
Most E14.5- to E16.5-day BAC-Col10a1-lacZ-neo transgenic embryos showed some X-gal staining in hair follicles of the forehead and snout, and of the epidermis of the tail, legs and shoulder, which disappears after birth. In the X-gal positive spots, staining was restricted to the basal layer of keratinocytes. Since these cells do not express type X collagen, the effect may be due to endogenous galactosidase activity, or a result of LacZ activation by other, Col10a1-unrelated regulatory elements located in the BAC clone.
In addition to Col10a1, the BAC clone RP23-192A7 contains coding regions of three additional genes Tspyl-1, Tspyl1-4 and Nt5dc1. Whether these genes are expressed under their endogenous promoters in the BAC transgenic mice is currently investigated. If they are expressed, they could affect the phenotype of the established transgenic mouse lines, but no phenotypic alteration in skeletal elements nor in other tissues have been observed in transgenic embryos, newborn or adults as compared to wildtype littermates.
The high intensity of X-gal staining under the control of the BAC-Col10a1 promoter allowed the visualization of discrete zones of Col10a1 expression in hypertrophic cartilage of anatomical structures not investigated so far in such detail and completeness, for example the skull or the inner and outer aspects of the thoracal and cervical vertebrae. The data presented in this study illustrate the superior potential of reporter gene expression analysis in combination with BAC recombineering technique as compared to conventional in situ hybridization analysis.
The robust and specific expression of the lacZ reporter gene under the control of BAC-Col10a1 environment furthermore shows that the placH+Col10a1-lacZ-frt-neo-frt targeting vector may be an extremely versatile vector for the targeted overexpression of other genes in hypertrophic chondrocytes of growth plates with high efficiency and specificity. Thus, the Col10a1-based BAC vector may be a useful tool to analyze the role of effector genes in skeletal development, endochondral ossification, and fracture callus formation.

bac
hypertrophic cartilage
homologous recombination
collagen x
transgenic mouse