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# PCPE-2 (procollagen C-proteinase enhancer-2): The non-identical twin of PCPE-1

Manon Napoli, Julien Bauer, Christelle Bonod, Sandrine Vadon-Le Goff, Catherine Moali

► To cite this version:

Manon Napoli, Julien Bauer, Christelle Bonod, Sandrine Vadon-Le Goff, Catherine Moali. PCPE-2 (procollagen C-proteinase enhancer-2): The non-identical twin of PCPE-1. Matrix Biology, 2024, 134, pp.59-78. 10.1016/j.matbio.2024.09.001. hal-04727512

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Submitted on 9 Oct 2024

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Review

PCPE-2 (procollagen C-proteinase enhancer-2):
The non-identical twin of PCPE-1

Manon Napoli, Julien Bauer, Christelle Bonod, Sandrine Vadon-Le Goff, Catherine Moali*

Université Claude Bernard Lyon 1, CNRS UMR 5305, Tissue Biology and Therapeutic Engineering Laboratory (LBTI), 69367 Lyon, France

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ARTICLE INFO

Keywords:

Metalloprotease

Inhibition

Collagen

Lipid metabolism

Inflammation

Cancer

A B S T R A C T

PCPE-2 was discovered at the beginning of this century, and was soon identified as a close homolog of PCPE-1 (procollagen C-proteinase enhancer 1). After the demonstration that it could also stimulate the proteolytic maturation of fibrillar procollagens by BMP-1/tolloid-like proteinases (BTPs), PCPE-2 did not attract much attention as it was thought to fulfill the same functions as PCPE-1 which was already well-described. However, the tissue distribution of PCPE-2 shows both common points and significant differences with PCPE-1, suggesting that their activities are not fully overlapping. Also, the recently established connections between PCPE-2 (gene name *PCOLCE2*) and several important diseases such as atherosclerosis, inflammatory diseases and cancer have highlighted the need for a thorough reappraisal of the *in vivo* roles of this regulatory protein. In this context, the recent finding that, while retaining the ability to bind fibrillar procollagens and to activate their C-terminal maturation, PCPE-2 can also bind BTPs and inhibit their activity has substantially extended its potential functions. In this review, we describe the current knowledge about PCPE-2 with a focus on collagen fibrillogenesis, lipid metabolism and inflammation, and discuss how we could further advance our understanding of PCPE-2-dependent biological processes.

Introduction

The first evidence of the existence of PCPE-2 was obtained inciden-
tally when Xu et al. identified a derived expressed sequence tag (EST) in
the primary open-angle glaucoma region of chromosome 3q21–24 [1].
Intraocular pressure being a validated risk factor for glaucoma, they
were looking for genes that could potentially lead to a defective extra-
cellular matrix in the trabecular meshwork of the eye and to impaired
aqueous humor filtration. This EST attracted their attention because of
its high similarity with two genes known to play major roles in collagen
fibrillogenesis, *BMP1* (encoding the bone morphogenetic protein-1 and

its splice variant mammalian tolloid, also known as mTLD) and *PCOLCE*
 encodes the procollagen C-proteinase enhancer-1 or PCPE-1). The
 sequence of the full cDNA corresponding to this EST showed that the
 highest sequence identity was with *PCOLCE* and the new gene was hence
 designated *PCOLCE2*. Beyond their overall 46% identity at the protein
 level (mature forms, Table 1), the two sequences also revealed a similar
 domain organization with 2 contiguous CUB (Complement, Uef, and
 BMP-1) domains, separated by a short linker (9 amino-acids) and fol-
 lowed by another longer linker (28–44 amino-acids) and a C-terminal
 NTR (netrin-like) domain [1] (Fig. 1a). The highest sequence conser-
vation between the two proteins lies in the CUB domains and is

**Abbreviations:** ADAMTS, A disintegrin and metalloproteinase with thrombospondin motifs; APOA1, Apolipoprotein a1; BMP, Bone morphogenetic protein; CAF, Cancer-associated fibroblast; DPC, Days post conception; ECM, Extracellular matrix; EMT, Epithelial to mesenchymal transition; EST, Expressed sequence tag; GTEx, Genotype tissue expression; HDL, High-density lipoprotein; HDL-C, HDL cholesterol; HDL-CE, HDL cholesteryl ester; HPA, Human protein atlas; IER, Intermittent energy restriction; IHC, Immunohisto-chemistry; LDLR, Low-density lipoprotein receptor; MDM, Monocyte-derived macrophage; MEF, Mouse embryonic fibroblast; mTLD, Mammalian tolloid; mTLL, Mammalian tolloid-like; NAFLD, Non-alcoholic fatty liver disease; NASH, Non-alcoholic steatohepatitis; NMR, Nuclear magnetic resonance; PCP, Procollagen C-proteinase; PCPE, Procollagen C-proteinase enhancer; PMA, Phorbol 12-myristate 13-acetate; pMPH, Peritoneal macrophages; PRM, Parallel reaction monitoring; PTM, Post-translational modification; RMSD, Root-mean square deviation; ROS, Reactive oxygen species; SAT, Subcutaneous adipose tissue; SFRP, Secreted frizzled related protein; SIRS, Severe inflammatory response syndrome; α-SMA, α-Smooth muscle actin; SNP, Single nucleotide polymorphism; SPR, Surface plasmon resonance; SR-BI, Scavenger receptor class B type I; TAM, tumor-associated macrophage; TCGA, The cancer genome atlas; TMSC, Tonsil mesenchymal stem cell; TPM, Transcripts per million; VAT, Visceral adipose tissue; WT, Wild-type.

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: catherine.moali@cnrs.fr (C. Moali).</footnote>
<caption>**Table 1**</caption>

Sequence identity and calculated isoelectric points of PCPE-1, PCPE-2 and their individual domains (after signal peptide removal). The sequence of the NTR domain is less conserved than the sequences of the CUB domains, yet the iso-electric points (pI) of the NTR domains only differ by 0.2 unit while those of CUB domains differ by almost 1 unit. % identities were computed with ClustalO (www.uniprot.org/align/) and theoretical isoelectric points with ProtParam (www.expasy.org/resources/protparam).

<table><thead><tr><td>Protein/Domain</td><td>% identity</td><td>pI</td></tr></thead><tbody><tr><td>PCPE-1 (45.5 kDa)</td><td>45.9</td><td>7.55</td></tr><tr><td>PCPE-2 (43.2 kDa)</td><td></td><td>8.67</td></tr><tr><td>CUB1 – PCPE-1</td><td>53.6</td><td>5.77</td></tr><tr><td>CUB1 – PCPE-2</td><td></td><td>6.75</td></tr><tr><td>CUB2 – PCPE-1</td><td>59.1</td><td>4.33</td></tr><tr><td>CUB2 – PCPE-2</td><td></td><td>5.28</td></tr><tr><td>NTR – PCPE-1</td><td>37.0</td><td>9.44</td></tr><tr><td>NTR – PCPE-2</td><td></td><td>9.66</td></tr></tbody></table>

somewhat lower for the NTR domains (Table 1). The disulfide pattern and the calcium-binding residues, first identified for PCPE-1, are also fully conserved [1,2] (Fig. 1a).

A few months after the publication of Xu et al., another group independently described ESTs from normal and osteoarthritic human cartilage with significant similarity to *PCOLCE* [4], confirming the existence of a homologous protein identical to the product of *PCOLCE2*. Then, the *Pcolce2* mouse gene was sequenced and mapped to chromosome 9 [5]. Mouse and human protein products have the exact same length after signal peptide removal (392 residues) but are significantly shorter than their *PCOLCE* homologs (respectively 424 and 444 residues for human and mouse PCPE-1 proteins) with missing stretches in the long linker and after the final cysteine of the NTR domain (Fig. 1a). Mouse and human *PCOLCE2* protein products share 89% identity and are well-conserved throughout the sequence including the long linker, in contrast with mouse and human PCPE-1 which differ significantly in length and composition in this particular sequence.

Although the protein derived from the new *PCOLCE2* gene seemed well expressed by trabecular meshwork cells, no coding sequence mutations were found in the glaucoma patient who was analyzed in the initial study [1] and the link with the disease could not be confirmed. However, the possible functional redundancy between the *PCOLCE* and *PCOLCE2* products was intriguing and prompted Steiglitz et al. to perform the first comparison of their tissue distribution, biochemical properties and ability to enhance procollagen C-terminal processing [6]. One of the most striking observations was the different subcellular localization of the two proteins. The *PCOLCE2* product, although secreted, could not be immuno-detected in the culture medium of the 293-EBNA cells used for recombinant expression but only within the cell layer. This was in agreement with the previous results obtained with trabecular meshwork cells [1] but in marked contrast with the behaviour of PCPE-1, that was mainly found in cell supernatants in the same conditions [1,6]. This property was suggested to be linked to a higher affinity for heparin and heparan-sulfate proteoglycans [6] and/or to the presence of a unique RGD sequence in the short linker of the human *PCOLCE2*-derived protein (Fig. 1A). This RGD sequence is conserved in most *PCOLCE2* sequences from mammalian and non-mammalian species with the notable exception of rat and mouse sequences where it is replaced by KGD. When present, the RGD sequence could promote cell attachment through integrin binding although this was never formally confirmed.

The main function of PCPE-1 is to stimulate the procollagen C-proteinase (PCP) activity of BMP-1 and of the related tolloid-like enzymes (mTLD, mTLL-1 and mTLL-2) [7,8], a major step in collagen fibrillogenesis. PCPE-1 is remarkably specific to this activity and, for example, does not affect the other functions of the proteinases [7]. Therefore, an important aspect of the initial characterization of the *PCOLCE2* product was to test its PCP enhancing activity. This was performed using

recombinant human procollagen II as substrate and BMP-1 or mTLL-1 as maturation enzymes. Similar enhancing activities for PCPE-1 and for the *PCOLCE2* product were observed in the conditions of the reaction [6] and this result led the authors to conclude that the product of *PCOLCE2* was also a functional procollagen C-proteinase enhancer and to name the new protein PCPE-2. This seminal result will be further discussed below, in light of recent findings indicating a more complex effect on procollagen processing, but the fact remains that the *PCOLCE2* gene and the PCPE-2 protein are systematically assigned to the collagen synthesis pathway. In the following sections, we will first review the available information on the physico-chemical properties and tissue distribution of PCPE-2, then discuss the current evidence suggesting that collagen synthesis may not be the most relevant pathway to describe PCPE-2 activity and, finally, propose new lines of research to further elucidate its biological functions.

## Physico-chemical properties and structures of PCPE-1 and PCPE-2

Since its initial discovery, PCPE-1 is known to be a glycoprotein capable of binding concanavalin A [9]. Its sequence actually contains two putative sites for N-linked glycosylations (Fig. 1a) but only the first one (asparagine 4 in the mature sequence) could be confirmed experimentally [10]. For PCPE-2, despite the presence of a similar potential site for N-linked glycosylations in the NTR domain (Fig. 1a), treatment with PNGase F did not lead to any increase in the electrophoretic mobility of the recombinant protein produced in 293-EBNA cells, suggesting that it was not used [6]. In contrast, the two proteins were shown to contain O-linked oligosaccharides and were affected by sialidase A, indicating the presence of sialyl residues [6]. The use of additional glycosidases suggested that sialylation was associated with galactosyl β1,3 N-acetyl-d-galactosamine (Galβ1,3GalNAc) O-linked cores in the case of the *PCOLCE2* product. The nature of the O-linkages was found to differ in PCPE-1 but could not be precisely identified. In a more recent study on human kidney samples [11], the presence of O-linkages was also evidenced on serine 284 of PCPE-1 and on threonine 270 of PCPE-2, both at the end of the long linkers. Phosphorylation sites are also reported for the two proteins at several sites spanning the three domains and the linkers [12]. However, the description of their respective PTM (post-translational modification) patterns is still incomplete and would be a valuable information to better map electrostatic charge distributions at their protein surface. Interestingly, based on sequence composition only, CUB domains are predicted to be more acidic in PCPE-1 than in PCPE-2 (Table 1). In contrast, the NTR domain appears highly basic with similar calculated isoelectric points (pI) around 9.5 for both PCPE-1 and -2. Overall, this gives a theoretical pI around physiological pH for full-length PCPE-1 and markedly more basic for full-length PCPE-2 (Table 1). Depending on the presence of specific PTMs, these values certainly differ in native proteins, as previously demonstrated by isoelectric focusing of human serum PCPE-1 which was identified as several isoforms with pIs between 5.3 and 6.8 [13].

At the structural level, there is presently no information available regarding PCPE-2 except the predicted fold computed by AlphaFold [14]. The AlphaFold scores are high for the three identified domains of the protein, suggesting that prediction is reliable, and it is interesting to compare these models with the X-ray or NMR (nuclear magnetic resonance) structures previously determined for PCPE-1 domains [15,16] (Fig. 1b). Calculated root-mean square deviation (RMSD) values for the structures of the CUB and NTR domains of the two proteins are lower than 1 Å showing that the high sequence conservation noted above is likely to translate into high structural conservation. Another level of functional significance is given by the distribution of charges at the surface of the protein domains (Fig. 1c). The CUB2 domains are characterized by a strong concentration of negative charges, specifically on one face, and the NTR domains by a strongly positive surface, in agreement with their respective pIs and with the high affinity of the NTR
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(caption on next page)
**Fig. 1.** Comparison of the sequences and structures of human PCPE-1 (UniProt Accession Number: Q15113) and human PCPE-2 (UniProt Accession Number: Q9UKZ9). (A) Sequence alignment of the mature proteins (after signal peptide removal) obtained with Uniprot ClustalO ("★" conserved residues; ":" strongly similar residues; "." weakly similar residues). The three main domains (CUB1, CUB2 and NTR) are highlighted in light yellow, green and grey respectively, the conserved cysteines in dark green and the residues involved in calcium binding in purple. Disulfide bonds are also shown. Bold residues in orange indicate putative N-glycosylation sites and bold residues in blue a potential integrin-binding (RGD) site, unique to PCPE-2. (B) Structural alignment of the domains of PCPE-1 (orange) and PCPE-2 (green). Individual domains were extracted from PDB structures 6FZV (for PCPE-1 CUB domains) and 1UAP (for PCPE-1 NTR domain) or modeled with AlphaFold2 (for PCPE-2). AlphaFold scores (predicted local distance difference test or pLDDT) for PCPE-2 were all superior to 92. Domains were superimposed and root-mean square deviations (RMSD) calculated using PyMOL [3] (calcium ions shown as pink spheres). (C) Comparison of the charge distribution for each domain of the two proteins on two opposite faces (the left panel shows the same orientation as in B). Surface representation colored according to electrostatic potential (red negative, blue positive).

domains for negatively-charged heparan sulfate proteoglycans [6, 17–20]. However, here again, there is no major difference between PCPE-1 and PCPE-2 with similar blue/red charge patterns on the three domains.

## PCPE-2 tissue distribution

### During development

The expression of the *Pcolce2* gene product during development was described in mice [5] and compared to *Pcolce* expression. By Northern blot, the two gene products displayed a similar temporal expression with a decrease between 7 and 11 dpc (days post conception) followed by a strong increase at 15 and 17 dpc [6,21]. Interestingly, *in situ* hybridization revealed prominent differences in spatial distribution, especially at 13.5 and 15.5 dpc. While *Pcolce2* expression was confined to the non-ossified areas of embryo cartilaginous structures (e.g. ribs, vertebrae, innominate bone) and was notably absent from regions undergoing ossification, *Pcolce* detection was more broadly associated with mesenchymal tissues and, in skeletal structures, with ossified regions. This distinct spatial distribution of the two gene products between non-ossified and ossified regions suggests specific and non-overlapping functions in the formation of bones. This finding could be linked to distinct roles of PCPE-1 and -2 in controlling BTP-dependent morphogen gradients and/or collagen assembly, as will be discussed below. Another study also reports that *Pcolce2* is expressed at 12.5 dpc in the thymic anlage of mouse embryos [22]. The gene is produced specifically by thymic epithelial cells and not by thymocytes but despite the critical role of epithelial cells in T cell production, this profile could not be linked to a role of *Pcolce2* in thymopoiesis [22].

### In adult tissues

#### Heart and vasculature

In the first articles describing PCPE-2 [1,4,6], Northern blot analyses of various human tissues were used to obtain preliminary information about its tissue distribution. In all cases, the transcript corresponding to *PCOLCE2* was found with the highest expression in the heart. Steiglitz et al. also used dot blot arrays to identify specific parts of the heart with high expression but it seemed equally well distributed between ventricles, atria, interventricular septum and apex [6]. They also found high expression in the aorta. This expression was specific to adult tissues because, in agreement with *in situ* hybridization, the expression in fetal heart was very low. More recent large-scale omics analyses confirmed these results. For example, on the Genotype Tissue Expression (GTEx) portal, the largest atlas of human gene expression data [23], the top tissues where *PCOLCE2* is most abundant, among a defined and non-exhaustive list, are different types of arteries (including the aorta) and the heart atrial appendage (Fig. 2).

The human heart and vasculature are also very strong in the PeptideAtlas database which compiles peptides detected in proteomic studies [25]. In addition, extensive quantitative proteomic characterizations of different anatomical regions of the human heart are available for male individuals aged 21–47 years [26] or 43–60 years [27]. Notably, the work of Doll et al. [26] shows that PCPE-2 is present in all 16 analyzed

heart regions with the highest levels in the aortic and mitral valves and the lowest levels in the right ventricle and ventricular septum. In the same study, BMP-1 was also expressed in the heart, with the highest abundance in the aortic valve, but it was generally found at lower levels in other regions and not detected in the atria and pulmonary vein. The analysis of different cardiac cell types also showed that PCPE-2 was more abundant in cardiac fibroblasts than in adipose fibroblasts, smooth muscle cells or endothelial cells (cardiomyocytes were not tested in this study). The high abundance of PCPE-2 in the aortic valve was also confirmed by a spatially and temporally resolved multi-omics analysis [28], which further demonstrated the specific expression of the transcript and protein in the fibrosa layer of the valve. This layer is rich in collagens and corresponds to the aortic side of the valve, as opposed to the spongiosa and ventricularis layers.

Finally, PeptideAtlas data reveal the presence of PCPE-2 in human plasma, confirming that it is a secreted protein that can be released in the circulation.

#### Adipose tissue

From the GTEx database, the subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) are also among the human adult tissues with the highest level of *PCOLCE2* expression (Fig. 2a). RNAseq data in Human Protein Atlas (HPA [29]) further indicate that *PCOLCE2* in adipose tissue is mostly provided by fibroblasts and adipocytes and that it is highly expressed in adipocytes from various origins (SAT, VAT, breast, skin). Adipose tissue expression of *Pcolce2* was also found by RT-qPCR in the VAT of male mice fed with a high fat/high cholesterol diet [30]. In a panel of 100 inbred strains of male and female mice placed on a high fat/high sucrose diet, *Pcolce2* mRNA abundance in SAT or VAT was correlated with percent fat mass and, in obese human subjects, with percent fat mass and body mass index [30]. In the same study, Xu et al. isolated cells from the stromal vascular fraction of murine SAT (in a *Ldlr*⁻/⁻ background) and differentiated them into adipocytes. During the differentiation process, *PCOLCE2* mRNA was found to gradually decrease and was <50% of its initial level in the fully differentiated state. These findings should be considered in light of the growing evidence that PCPE-2 is an important player in regulating lipid metabolism, as will be discussed in the dedicated section below.

#### Reproductive organs and endocrine system

Prominent signals in PeptideAtlas are also found for the pituitary gland, a major structure for hormone production regulating reproduction, growth and metabolism. Interestingly, the pituitary gland and several pituitary-dependent components of the female reproductive organs (vagina, cervix, mammary gland) are among the tissues with highest *PCOLCE2* expression in GTEx data (Fig. 2a). In line with these results, *PCOLCE2* expression in the pituitary gland was also detected by Northern blot in early studies [6]. More recently, Banerjee et al. observed that *PCOLCE2* was specifically enriched in the pituitary gland compared to fifteen other neuroendocrine glands and brain regions [31]. For example, the hypothalamus and adrenal gland, although belonging to the same endocrine axis, comparatively show very limited *PCOLCE2* expression [6,31]. The pituitary gland itself is composed of two distinct lobes. The posterior lobe is a direct extension of the hypothalamus while the anterior lobe is composed of different endocrine cells secreting
A

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B

<figure><img src="image_8.png" /><figcaption>**Fig. 2.** PCPE-2 expression in the adult human body. (A) Twelve tissues with the highest *PCOLCE2* RNA abundance compiled from GTEx transcriptomic data and comparison with *BMP1* and *PCOLCE* levels. TPM means transcripts per million. (B) Overview of expression sites for PCPE-2 (RNA or protein level) described in the literature (see text for references). Major expression sites are highlighted in bold. Created in BioRender [24].</figcaption></figure>
specific hormones. Using quantitative proteomics, Banerjee et al. showed that PCPE-2 was one of the most discriminating proteins between the anterior and posterior lobes of the healthy pituitary gland. This specificity profile was confirmed by targeted proteomics (PRM) and is notably similar to the expression profile of prolactin and growth hormone [31], two known substrates of BMP-1 [32] (see the section entitled “Other potential roles of PCPE-2 linked to BMP-1 inhibition” for more detail). *PCOLCE2* was then confirmed, by single cell RNA sequencing, to be expressed by hormone-producing cells of the pituitary gland and identified as a top expressed gene in corticotroph cells [33].

Beyond prolactin, which stimulates milk production during pregnancy and lactation, the anterior pituitary also produces FSH (follicle-stimulating hormone) and LH (luteinizing hormone) which regulate the development of ovarian follicles in females and of spermatocytes in males. Furthermore, the posterior lobe secretes oxytocin which is involved in milk production and uterine contractions. Thus, it is interesting to note that PCPE-2 is widely expressed in female organs such as the mammary gland, vagina and uterus [5] (Fig. 2). Low expression is also found in ovaries and fallopian tubes, at the RNA and protein levels [34]. Strikingly, *PCOLCE2* was found to be increased >26-fold upon culturing porcine ovarian granulosa cells for up to 6 days [35]. It was one of the most upregulated ECM genes in this context and, based on the prominent role of granulosa cells in folliculogenesis and oogenesis, this finding may lead to novel options for the optimization of assisted reproduction techniques [35]. Finally, as previously qualitatively described [1,4,6] and based on PeptideAtlas and HPA data, *PCOLCE2* is highly expressed in placenta both at the RNA and protein levels. In the HPA transcriptomic data, its abundance is just slightly lower than in the heart and adipose tissues. The HPA database also reports a strong cytoplasmic positivity in trophoblastic cells, both by single cell RNAseq and immunochemistry.

### Collagen-rich tissues

PCPE-2 was originally identified in the trabecular meshwork of the eye and its expression was confirmed in trabecular meshwork cells at the RNA and protein levels [1]. In these cells, PCPE-2 was detected in two forms, the canonical full-length protein (around 50 kDa) and a proteolytic fragment of approximately 17 kDa, the origin of which is still unclear. In the eye, *Pcolce2* was also detected in the mouse cornea by RT-qPCR, both in the corneal stroma and epithelium [36]. However, the protein expression could not be confirmed by Western blot, probably because of its low tissue concentration [37]. In contrast, in the cochlea which is part of the inner ear, PCPE-2 was detected by Western blot in mice with a peak of expression seven days after birth and subsequent decrease until day 30 [38]. In this case, *Pcolce2* was specifically expressed by the sensory cells of the cochlea called the hair cells.

In one of the early studies which have led to its identification [4], *PCOLCE2* mRNA was found in adult articular cartilage, at a relatively high level comparable to the heart and in line with its strong detection in embryonic cartilaginous structures [6]. The PCPE-2 protein product was also identified in articular cartilage vesicles [39]. In the intervertebral disc, made of fibrocartilage, proteomic analyses have revealed that it is also present and downregulated in the inner disc of aged human individuals compared to young individuals [40]. In contrast, there was no significant difference in the murine intervertebral disc between compartments, spine levels (lumbar versus tail) or genders [41].

PCPE-2 is also present in skin, among the 10,701 proteins described in the Human Skin Atlas [42]. Its presence is detected at similar levels across the three skin layers: epidermis, dermis, and hypodermis. All the analyzed cell types (keratinocytes, fibroblasts, dendritic cells, endothelial cells, macrophages, mast cells and melanocytes) seem to express PCPE-2 with a slight enrichment in endothelial and mast cells. This profile differs significantly from that of PCPE-1, which is substantially more abundant in the dermis than in other skin layers and in fibroblasts relatively to other cell types. PCPE-1 is also much more abundant in the dermis than PCPE-2 but, interestingly, *PCOLCE2* emerges as one of the

most differentially expressed genes between fibroblasts of the papillary and reticular dermis [43,44]. The preferential expression in the reticular dermis probably reflects the different functions of the two sub-layers which is now well-established [43]. Sexual dimorphism was also observed regarding the expression of *PCOLCE2* in skin with higher expression in females compared to males for both sun-exposed and sun-protected skin [45]. In mice, *Pcolce2* is among the 514 down-regulated genes in photoaged back skin in a chronic low-dose UV irradiation model [46].

### Other tissues

Northern blot assays highlighted some moderate expression of *PCOLCE2* in multiple additional organs such as lung, liver, kidney, skeletal muscle, bladder, esophagus and trachea [1,4,6]. This is confirmed for most tissues in the GTEx dataset (see Fig. 2a for bladder and esophagus). For lung, PCPE-2 expression was confirmed at the protein level in mice and it was observed to be enriched in the insoluble fraction where most core structural ECM proteins are found [47]. There was no difference in protein abundance nor solubility profile between the lungs of old (24 months) and young (3 months) mice [48]. Among the 30 cell types which could be identified by scRNASeq in mouse lung, *Pcolce2* was significantly enriched in capillary endothelial cells, interstitial fibroblasts and lipofibroblasts [48]. In human skeletal muscle, *PCOLCE2* was used to define a sub-cluster of fibro-adipogenic progenitor cells [49] which are muscle interstitial mesenchymal cells providing a supportive environment for muscle stem cells and involved in muscle regeneration. Finally, PCPE-2 was identified as a component of the O-glycoproteome in human kidney samples [11] confirming its expression in this tissue. *PCOLCE2* was also shown to be a novel marker for podocytes, specialized cells critically involved in maintaining the selective filtration barrier in kidney, based on its correlation with previously known markers of this cell type [50]. This podocyte specificity of PCPE-2 is in agreement with the staining pattern shown in the HPA portal.

In summary, PCPE-2 is co-expressed with PCPE-1 and BMP-1 in most of the tissues listed above (Fig. 2a). However, the relative levels of the three proteins can vary substantially and PCPE-2 shows a distinct and unique expression pattern at the sub-tissue level with specific enrichment in defined functional structures or cell populations. The distinct expression profiles of PCPE-1 and PCPE-2 also suggest that their biological functions are not fully overlapping, as will be further discussed in the following sections.

### Biological functions and related diseases

In 2007, in an attempt to evaluate PCPE-2 involvement in thymopoiesis, Heinzel and Bleul generated the first (and still unique) mouse model where the *Pcolce2* gene is constitutively inactivated [22]. At the time, their study was motivated by the demonstration of the regulation of *Pcolce2* expression by a transcription factor, Foxn1, also controlling thymic epithelial cell differentiation. *Pcolce2*-deficient mice were viable and fertile, showed no obvious developmental or phenotypic abnormalities and, more unexpectedly, the absence of *Pcolce2* had no impact on thymus development. The authors did not characterize the mice further but the absence of severe defects in basal conditions was confirmed by subsequent studies [19,51,52].

Thus, despite a rather wide tissue expression, PCPE-2 seemed to be dispensable for normal development and its biological functions remained unclear. Now, and despite the relatively rare functional studies focusing on PCPE-2, some progress has been made to better delineate its roles. A remarkable conclusion that is now emerging is that the functions of PCPE-2 are not fully described by its initial characterization as a procollagen C-proteinase enhancer. Especially, its involvement in lipid metabolism, inflammation or cancer progression is now supported by a growing body of evidence. Our current understanding of these various functions is reviewed below.
## Role of PCPE-2 in collagen fibrillogenesis, tissue repair and fibrosis

### In vitro observations

As mentioned above and indicated by its name, PCPE-2 was for many years solely described by homology with PCPE-1, a well-known regulator of fibrillar collagen biosynthesis [7]. Fibrillar collagens I, II, III, V and XI are produced by cells as precursor molecules, procollagens, which need to undergo proteolytic maturations to form fibrils in the extracellular environment. The removal of procollagen N- and C-proteptides by dedicated proteases leads to a strong decrease in collagen solubility and to enhanced lateral aggregation that are essential for fibril assembly [53,54]. On the N-terminal side, it is mainly ADAMTS (a

disintegrin and metalloproteinase with thrombospondin motifs) 2, 3 and 14 which cleave the N-propeptide [55] whereas, on the C-terminal side, enzymes of the BTP (BMP-1/tolloid-like proteinases) family are in charge of C-propeptide removal [8]. The BTP family is composed of four members (BMP-1, mTLD, mTLL-1 and mTLL-2) [8] but mTLL-2 is usually not considered as a procollagen C-proteinase because of its very low activity on procollagens [56]. PCPE-1 has no proteolytic activity *per se* but stimulates the C-terminal procollagen maturation by BTPs in a very efficient and specific manner, through a direct and tight binding to the procollagen C-propeptides [7].

In the first study that described the procollagen C-proteinase enhancing activity of PCPE-2, Steiglitz et al. produced a recombinant

<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Dual role of PCPE-2 in the control of BMP-1 activities and comparison with PCPE-1. Diagram of cleavage activity (normalized to the activity of BMP-1 alone) for procollagens I-III (A-B) or other BMP-1 substrates (C-D) in the presence of increasing concentrations of PCPE-1 (A, C) or PCPE-2 (B, D). (A-B) In a typical procollagen cleavage assay, the substrate concentration far exceeds the BMP-1 concentration while the PCPE concentration is in the same range as the procollagen concentration. Therefore, PCPE-1 and -2 preferentially bind the procollagen and enhance its cleavage by BMP-1 (A, B). When PCPE-2 concentration increases, binding to BMP-1 becomes significant (due to similar $K_D$s of PCPE-2 for procollagen and BMP-1) and blocks its catalytic activity, thereby leading to the inhibition of the reaction (B). In contrast, PCPE-1 binding to BMP-1 remains low or unproductive in the relevant concentration range and procollagen processing continues to be stimulated. (C-D) For other BMP-1 substrates, PCPE-1 has no impact on their cleavage (C) while PCPE-2 behaves as a potent inhibitor (D).</figcaption></figure>
form of the protein and showed, using co-immunoprecipitation and electron microscopy, that it was able to bind to (pro)collagens I and II, similarly to PCPE-1 [6]. This property was later extended to procollagen III [19]. Importantly, the affinities of both PCPE-1 and PCPE-2 for the C-terminal portion of procollagen III are in the same range (dissociation constants of 2.2 and 11 nM respectively, measured by surface plasmon resonance (SPR)) [19]. As concomitant binding on procollagens is precluded [19], it is also clear that both PCPEs bind to overlapping (and probably identical) sites. The founding work of Steiglitz et al. also reported that, like PCPE-1, PCPE-2 had the capacity to efficiently enhance the processing of recombinant procollagen II by BMP-1 or mTLL-1 [6]. Likewise, the *in vitro* cleavage of procollagen I in concentrated fibroblast supernatant was found to be boosted by the addition of PCPE-2 [57]. However, this was not the case in another study, which reported that procollagen I processing was not increased when PCPE-2 was incubated with the medium of wild-type fibroblasts nor was it reduced in the conditioned medium of fibroblasts extracted from *Pcolce2*-deficient mice [19].

These discrepancies prompted us to take a closer look at the effect of PCPE-2 on the C-terminal cleavage of procollagens [19]. Using recombinant mini-procollagens I-III and the BMP-1 protease, we actually confirmed a consistent enhancing activity of PCPE-2, although substantially lower than that of PCPE-1. However, PCPE-2 did not behave like a canonical procollagen C-proteinase enhancer. When its concentration increased, the stimulation was progressively abolished and replaced by the inhibition of procollagen cleavage (Fig. 3a-b). This dual activity also occurred when mTLD and mTLL-1 were used as procollagen proteinases.

Also, unexpectedly and in contrast to PCPE-1, which has no effect on other BTP substrates [7] (Fig. 3c), PCPE-2 was found to inhibit the cleavage of a long (and non-exhaustive) list of BMP-1 substrates: thrombospondin-1, betaglycan, perlecan/endorepellin, chordin, procollagen V (N-terminal cleavage) and LDL receptor [19]. For these substrates, only inhibition was observed, in a PCPE-2 concentration-dependent manner (Fig. 3d), but no initial stimulation like for procollagens I-III. Mechanistically, the inhibitory effect is mediated by a direct and strong interaction between the regulatory protein and the protease ($K_D \sim 8$ nM), involving the domains of PCPE-2 which are also involved in procollagen binding (CUB1 and CUB2) [16,58]. Thus, the interactions of PCPE-2 with BMP-1 and procollagen are mutually exclusive, explaining the biphasic activity depicted in Fig. 3b. At low concentrations, PCPE-2 preferentially binds procollagen, the latter being more concentrated than the protease in the cleavage assay, and then induces the activation of procollagen cleavage by a mechanism similar to the one described for PCPE-1, involving the local disruption of the procollagen trimeric structure [16]. In contrast, at higher concentrations, when most binding sites on procollagen are occupied, the free remaining PCPE-2 binds the protease and inhibits its activity, thereby hindering procollagen cleavage. The net effect on procollagen processing (absence of effect, stimulation, inhibition) therefore directly depends on the relative concentrations of the three partners (procollagen, protease and PCPE-2). The main challenge is to extrapolate this complex regulatory mechanism to *in vivo* conditions and to predict the outcome of the regulatory activities of PCPE-1 and −2 in the complex tissue environment. Interestingly, we have observed that when both proteins are present in equimolar concentrations, the enhancing effect of PCPE-1 is blunted and the inhibitory effect of PCPE-2 is enhanced [19]. This can be explained by the occupancy of procollagen binding sites by PCPE-1, which indirectly promotes the interaction of PCPE-2 with BMP-1 and hence the inhibition. However, the absence of obvious abnormalities in the collagen fibrils of *Pcolce2*-deficient mice [19] suggests that, at least during development and in homeostasis conditions, the effect of PCPE-2 on procollagen maturation is moderate or ineffective, as will be more extensively discussed in the section entitled "*In vivo* observations".

## PCPE-2 in fibroblasts

Most of the studies characterizing PCPE-2 in cells have been conducted in fibroblasts (or fibroblast-like cells), the main collagen-producing cells and the main cell type for the production of the homologous PCPE-1 protein. As noted above, a first intriguing observation was that the two proteins are not found in the same cellular compartment in cultures of mouse embryonic and human trabecular meshwork fibroblasts. PCPE-2 was detected in the cell extracts (including the intracellular cell content, surface-bound proteins and extracellular matrix) whereas PCPE-1 was more abundant in the cell supernatant [1,59]. The impact of this apparent sequestration of PCPE-2 in the cell layer, which may limit its ability to diffuse and reach the procollagens or the BTPs, has never been evaluated and it is not known if it could be a way to modulate its activity. On the other hand, this could also lead to potentiating effects as confinement in defined extracellular microenvironments (e.g. fibronectin- or heparan-sulfate-rich niches) is often seen in the ECM and can be used as a concentration/scaffolding mechanism to promote specific activities [17,20,60].

Another parameter to better understand PCPE-2 function in fibroblasts and its cross-talk with PCPE-1 is the relative amounts of the two proteins. Strikingly, even if fibroblasts are the main PCPE-2 producing cells, as reported by the HPA database where *PCOLCE2* is assigned to the fibroblast cell cluster, it is produced in low amounts compared to *PCOLCE*. In both adult human and mouse dermal fibroblasts, the *PCOLCE2* mRNA quantified by qRT-PCR was found to be at least 40-fold less abundant than *PCOLCE* mRNA [19]. This was also the case in fetal human dermal fibroblasts and in amniotic fluid mesenchymal cells where *PCOLCE2* was reported to be 50-fold and 300-fold less expressed than *PCOLCE* respectively [61]. This difference is slightly mitigated when fetal and adult skin fibroblasts are cultivated on a rigid matrix but remains substantial [62]. In mouse skin, both genes are less abundant in dermal papilla fibroblasts, residing at the base of hair follicles, versus pan-dermal fibroblasts [63] while, in human skin, only *PCOLCE2* can differentiate papillary fibroblasts found in upper skin dermis from reticular fibroblasts found in deeper layers [43], also suggesting different functions or mechanisms of action.

This dissimilarity in subcellular localization and in amounts of gene products could explain that, while PCPE-1 is clearly linked to increased collagen synthesis and fibrosis [7], it is not the case for PCPE-2. There is indeed no difference in the extent of procollagen processing between wild-type and *Pcolce2*-deficient skin fibroblasts [19]. In contrast, the lack of *Pcolce* in mouse corneal fibroblasts (keratocytes) slows down procollagen processing in cell supernatants [37]. Also, contrary to what might be expected for a protein involved in collagen synthesis, *PCOLCE2* expression is not increased upon differentiation of fibroblasts into myofibroblasts. Instead, *Pcolce2* was one of the most downregulated genes in α-SMA- (α-smooth muscle actin) positive myofibroblasts extracted from mouse skin wounds compared to α-SMA-negative fibroblasts [64]. In systemic sclerosis skin, it was found by single cell RNA sequencing that a *PCOLCE2*+ subpopulation of fibroblasts with high *SFRP2* (secreted frizzled related protein 2) expression rather appeared as progenitors in the trajectory of myofibroblasts [65]. Similarly, in kidney, scar-forming myofibroblasts express lower levels of *PCOLCE2* than “progenitor” non-activated fibroblasts [66]. These data indicate that *PCOLCE2* is rather a marker of fibroblasts with progenitor potential than of fibroblasts with high ECM synthesis capacity. Together, it seems that PCPE-1 and PCPE-2 fulfill different functions in fibroblasts, collagen-dependent or -independent respectively. We will describe in the section about “Other potential roles of PCPE-2 linked to BMP-1 inhibition”, how novel clues about the functions of PCPE-2 could potentially be found in the regulation of other BTP activities.

## *In vivo* observations

In line with previous observations, careful examination of *Pcolce2*-deficient mice showed that the absence of the gene product had no major impact on the homeostasis of collagen-rich tissues. No alteration of the
organization, morphology or number of collagen fibrils were observed in the skin, heart and tendons of these mice [19,51]. The structure and function of the heart or the tibia length were also preserved in deficient mice [51], as were skin mechanical properties [19]. This is in marked contrast with *Pcolce* deficiency which leads to highly dysmorphic collagen fibrils in tendons and bones and to altered long bone geometries and mechanical properties in male mice [59]. Importantly, it was also checked that there is no compensation between the two proteins. Mouse embryonic fibroblasts (MEFs) from *Pcolce*⁻/⁻ mice actually produce the same amount of PCPE-2 as wild-type mice and the skin of *Pcolce2*⁻/⁻ mice shows no significant difference in *Pcolce* RNA level when compared to the skin of wild-type mice [19,59]. However, sex-specific effects cannot be excluded as a slight diminution of *Pcolce2* mRNA was reported in the heart of *Pcolce*⁻/⁻ female mice but not in male mice [67].

If PCPE-2 is not crucial for proper collagen assembly in homeostasis conditions, in line with the moderate enhancement of procollagen processing observed *in vitro* [19], it could still play a major role during tissue repair when collagen synthesis is known to increase dramatically. This also raises the question of whether PCPE-2 could be a marker of fibrogenic or fibrotic conditions like BMP-1 and PCPE-1 [7,8,68]. Current knowledge on the role and expression of PCPE-2 in tissue repair and fibrosis is described below.

Since the heart is the organ with the highest level of PCPE-2, its involvement in cardiac repair has been of particular interest. In this field, a pioneer study was published by Baicu et al. [51], who studied the effect of the absence of *Pcolce2* gene in a mouse model of chronic pressure overload leading to myocardial fibrosis. When subjected to transverse aortic constriction (TAC), *Pcolce2*-deficient mice have reduced myocardial stiffening compared to wild-type (WT) mice. They also have reduced collagen deposition, with an increase in collagen volume fraction (measured by picrosirius red staining) of 2-fold instead of 6-fold for WT mice. This however was not accompanied by an increase in soluble unprocessed collagen. These data are intriguing and would need to be re-evaluated, in light of the new results showing the complex role of PCPE-2 in procollagen processing, to know if they reflect the direct involvement of the regulatory protein in this process or another indirect mechanism (e.g. through the regulation of lipid metabolism or inflammation, as will be described in the dedicated sections below). This is also necessary because, in another model of pressure overload induced pharmacologically [69], cardiac fibrosis was accompanied by an increase in *Pcolce* and *Bmp1* expression but *Pcolce2* remained unchanged. Increased *PCOLCE2* expression was however also reported in dilated cardiomyopathy (DCM), another frequent cause of myocardial fibrosis, both in patients and in a mouse model [70,71], and *PCOLCE2* has been included in a 27 gene-set signature for this pathology [70]. PCPE-2 was also analyzed in patients suffering from aortic stenosis induced by calcific aortic valve disease [28] and found to be one of the most enriched proteins in the “fibrotic” regions of the aortic valve. However, closer inspection reveals that PCPE-2 expression is confined to the fibrosa layer of the valve where collagens are concomitantly diminished in diseased samples [28].

In other organs, fibrotic diseases seem mostly associated with decreasing levels of PCPE-2. One exception is lumbar spinal canal stenosis, a disease caused by the thickening of the ligamenta flava in the vertebral columna, where PCPE-2 was quantified as increased (x 4.6) by mass spectrometry, together with fibronectin and proteins linked to chondrogenesis [72]. In contrast, in the lung, *PCOLCE2* downregulation was reported in patients with idiopathic pulmonary fibrosis in three different studies [73–75]. This downregulation was also detected in a mouse model of fibrosis induced by bleomycin treatment, both at the gene [76] and protein levels [47]. In the liver, it is also very notable that *PCOLCE2* was a common differentially expressed gene in four transcriptomic datasets collected from patients with liver fibrosis of different etiologies (hepatitis B or C, nonalcoholic fatty liver disease NAFLD) [77]. It was one of only four downregulated genes among 105 common

differentially expressed genes, also including fibrillar collagens, which were upregulated. *PCOLCE2* expression was also reported to be lower in NASH (non-alcoholic steatohepatitis, a more advanced form of NAFLD) patients with high fibrosis scores compared to control subjects [78], although the observed trend did not reach significance with the small number of patients analyzed. Similarly, it was one of the most down-regulated genes in a mouse model of hepatic steatosis induced by the lack of *Igf2* (insulin-like growth factor-II) [79]. As will be discussed in the section on the “Role of PCPE-2 in lipid metabolism and metabolic diseases”, the strong negative correlation between *PCOLCE2* and liver disease could also reflect the recently recognized involvement of PCPE-2 in lipid-related pathways.

Interesting data were also collected in external organs such as cornea and skin. For example, in a mouse model of corneal scarring induced by penetrating keratectomy, *Pcolce2* expression was reported to be decreased throughout the healing process at four different time points between 7 and 28 days, both in the epithelial and stromal layers [36]. This was in contrast to *Pcolce* and *Bmp1* genes, which were clearly upregulated after wounding. To our knowledge, no similar study has been performed in skin but some data report a dysregulation of *PCOLCE2* in Dupuytren’s disease, a fibrotic skin disease of the hand characterized by two stages called nodule and cord. In a large meta-analysis, *PCOLCE2* was found to be two-fold less expressed in diseased tissue than in normal tissue [80] but, in another study, it was 4-fold more abundant in the more advanced cord stage compared to nodule, also at the protein level [81]. Collagen I expression was inversely lower in the cord than in the nodule. Likewise, *PCOLCE2* was found downregulated (by 2.75-fold) in the dermal fibroblasts of a patient suffering from a severe fibrotic disorder of unknown etiology characterized by subcutaneous nodules [82].

Overall, published work on fibrotic diseases seems to indicate that the correlation between PCPE-2 abundance and collagen deposition is much less strong than for PCPE-1 and could even be inverse. Again, this suggests that the main role of PCPE-2 in these diseases is not in collagen maturation but in yet undefined functions. In the next sections, we will describe the novel functions that were recently attributed to PCPE-2 and could shed light on previous observations.

## Other potential roles of PCPE-2 linked to BMP-1 inhibition

The identification of PCPE-2 as an endogenous inhibitor of BMP-1 and related proteases (see section entitled “In vitro observations”) was an important breakthrough because it gave new insights not only into possible additional functions of PCPE-2 but also into the regulation of BTPs. It is indeed quite unusual for proteases with such a broad range of activities to lack a regulatory mechanism which can stop the potential deleterious effects of uncontrolled proteolysis. It is now clear that an endogenous protein inhibitor with nanomolar potency [19], PCPE-2, can contribute to fine-tune BTP-dependent proteolysis and counter-balance the numerous proteins that have the ability to enhance BTP activity (PCPE-1, periostin, twisted gastrulation, ONT-1, WFIKKN1, fibronectin [8]). Another endogenous inhibitor of BMP-1 was shown to be α2-macroglobulin [83] but it lacks the necessary specificity that warrants tight spatio-temporal regulation. Therefore, the discovery of PCPE-2 as the first specific endogenous inhibitor of human BTPs has broad potential consequences to describe their respective functions and raises a number of new questions.

First, the molecular mechanism by which PCPE-2 exerts its inhibitory activity on BMP-1 is not yet fully understood. It is already clear that PCPE-2 does not bind directly in the catalytic site of the protease and does not act as a competitive inhibitor [19]. In addition, the non-catalytic domains of BMP-1 are also needed for the inhibition [19], suggesting an allosteric mode of inhibition where PCPE-2 binding occurs through at least two interaction sites, one located in the non-catalytic domains and the other in the catalytic domain at a distant site from the catalytic pocket. However, the exact mechanism by which PCPE-2
can inhibit the BTPs without competing with substrate binding in the catalytic site, the most common mechanism of protease inhibition [84], remains to be characterized.

Another point that has not been investigated yet is the consequences of this inhibitory mechanism *in vivo*. Based on Fig. 2a, PCPE-2 could be synthesized in amounts similar to BMP-1 or even slightly superior in various tissues. This suggests that inhibition could play a role in regulating BMP-1 functions even in homeostasis conditions. BMP-1 and tolloid enzymes have pleiotropic functions, extending from the regulation of extracellular matrix assembly to the mineralization of hard tissues, TGF-β and BMP activation, muscle growth, angiogenesis, lipid metabolism, hormonal regulation and nerve conduction in the peripheral nervous system (Table 2). *In vitro*, the processing of a wide variety of BTP substrates with diverse functions, including thrombospondin-1, betaglycan, perlecan/endorepellin, chordin, procollagen V N-propeptide and LDL receptor, was already confirmed to be efficiently inhibited by PCPE-2 [19]. Interestingly, some hints were already obtained that the co-expression of the BTPs and their inhibitor is indeed biologically relevant as TGFB and BMP2 expression was downregulated in *Pcolce2-deficient* mice [19]. Increasing the bioavailability and activity of these growth factors is a major role of BTPs [85–87] and their down-regulation suggests a feedback compensatory mechanism in the absence of PCPE-2. In this context, it is also interesting to remind that PCOLCE2 cellular expression is often associated with progenitor potential (see

above), suggesting a role in cell differentiation and in morphogenesis. The possible link between PCPE-2 and progenitor cell differentiation was also exemplified recently in a mouse model of cochlea damage [38]. In this work, the authors showed that overexpression of *Pcolce2* by adeno-associated virus (AAV) transduction could promote the re-entry of progenitor Lgr5+ supporting cells into cell cycle and their differentiation into hair cells, which are sensory cells critical for normal hearing. This effect required the concomitant activation of the Wnt signaling pathway, suggesting the potentiation of this pathway by PCPE-2 [38]. However, for all the growth factors mentioned (TGF-βs, BMPs, Wnts), the direct link between PCOLCE2 expression and *in vivo* BTP inhibition remains to be established.

In addition to these possible roles in signaling, there is also evidence that PCPE-2 regulates the functions of BTPs in lipid metabolism as will be described in the next section. New hypotheses regarding possible PCPE-2 functions in angiogenesis, muscle growth, hormonal regulation or clustering of sodium channels in the peripheral nervous system can also be tested based on the known roles of BTPs (Table 2). Moreover, the possible modifications of the protease:inhibitor ratio under challenge or in disease conditions will also be important to analyze in appropriate biological settings. Understanding BTP regulation by PCPE-2 *in vivo* thus opens an entirely new field of research.

<caption>**Table 2**</caption>

Known substrates of BMP-1/tolloid-like proteinases and functions expected to be affected by PCPE-2. # indicates substrates for which PCPE-2 was already shown to inhibit *in vitro* cleavage [19] (see Fig. 3b for the specific case of procollagens I-III involving a complex profile of mixed inhibition and activation).

<table><thead><tr><th></th><th></th></tr></thead><tbody><tr><td><strong>Biological functions</strong></td><td></td></tr><tr><td><strong>Collagen and elastin assembly</strong></td><td></td></tr><tr><td>- Procollagen I (α1, α2)<sup>#</sup>, Procollagen II (α1)<sup>#</sup>, Procollagen III (α1)<sup>#</sup>, Procollagen V (α1<sup>#</sup>, α2, α3) and Procollagen XI (α1, α2)</td><td>[19,88–91]</td></tr><tr><td>- Collagen VI (α3)</td><td>[92]</td></tr><tr><td>- Small leucine-rich proteoglycans (Biglycan, Osteoglycin/mimecan, Decorin)</td><td>[93–95]</td></tr><tr><td>- Thrombospondin-1<sup>#</sup></td><td>[19,85]</td></tr><tr><td>- Lysyl oxidases (LOX, LOXL)</td><td>[96,97]</td></tr><tr><td><strong>Basement membrane assembly</strong></td><td></td></tr><tr><td>- Procollagen VII</td><td>[98]</td></tr><tr><td>- Laminin 332</td><td>[99,100]</td></tr><tr><td><strong>Mineralization of bones and teeth</strong></td><td></td></tr><tr><td>- Dentin matrix protein-1 (DMP-1)</td><td>[101]</td></tr><tr><td>- Dentin sialophosphoprotein (DSPP)</td><td>[102]</td></tr><tr><td><strong>TGF-β / BMP signaling</strong></td><td></td></tr><tr><td>- Latent TGF-β binding protein 1 (LTBP-1), Betaglycan (TGF-β receptor III)<sup>#</sup>, CD109, Thrombospondin-1<sup>#</sup></td><td>[19,85,87,103]</td></tr><tr><td>- Chordin<sup>#</sup>, BMP-11/GDF-11</td><td>[19,56,104]</td></tr><tr><td><strong>Muscle growth</strong></td><td></td></tr><tr><td>- Myostatin/GDF-8, WFIKKN1</td><td>[105,106]</td></tr><tr><td><strong>Angiogenesis</strong></td><td></td></tr><tr><td>- Perlecan/endorepellin (HSPG2)<sup>#</sup></td><td>[107]</td></tr><tr><td>- Prolactin, Growth hormone/somatostatin</td><td>[32]</td></tr><tr><td>- Thrombospondin-1<sup>#</sup></td><td>[19,85]</td></tr><tr><td>- Angiopoietin-like protein 2 (ANGPTL2)</td><td>[108]</td></tr><tr><td><strong>Lipid metabolism</strong></td><td></td></tr><tr><td>- Apolipoprotein A1</td><td>[109]</td></tr><tr><td>- LDL receptor<sup>#</sup></td><td>[19,110]</td></tr><tr><td>- Angiopoietin-like protein 2 (ANGPTL2)</td><td>[108]</td></tr><tr><td><strong>Hormones and adipokines</strong></td><td></td></tr><tr><td>- Prolactin, Growth hormone/somatostatin</td><td>[32]</td></tr><tr><td>- Insulin-like growth factor binding protein 3</td><td>[111]</td></tr><tr><td>- Collagen VI (α3)/endotrophin</td><td>[92]</td></tr><tr><td><strong>Peripheral nervous system development</strong></td><td></td></tr><tr><td>- Gliomedin</td><td>[112]</td></tr></tbody></table>
## Role of PCPE-2 in lipid metabolism and metabolic diseases

As highlighted above, *PCOLCE2* is highly expressed in the subcutaneous and visceral adipose tissues (Fig. 2) and is correlated to adipose mass in both humans and mice [30]. However, it is only recently that the regulation of lipid metabolism was identified as a prominent biological function of PCPE-2. More specifically, it seems to be critical for lipid transport, a complex pathway involving different types of lipoproteins (e.g. high-density lipoprotein and low-density lipoprotein, HDL and LDL respectively) found in the circulation and carrying dietary lipids, lipids synthesized in the liver or excess cholesterol from peripheral tissues. Important players in this pathway are apolipoproteins, such as ApoA1 (Apolipoprotein A1) in HDL and ApoB100 in LDL, which regulate both the structural integrity of lipoproteins and their metabolic function by facilitating their recognition by receptors or enzymes [113]. Moreover, various cellular receptors such as LDLR (low-density lipoprotein receptor) and SR-BI (scavenger receptor class B type I, a major receptor for HDL) play crucial roles in recognizing lipoproteins to promote lipid cellular influx and efflux. According to recent work, PCPE-2 could be involved in the control of the activity of several of these players with a direct impact on lipid metabolism and metabolic diseases.

First, in metabolic diseases, *PCOLCE2* was found to be down-regulated in the pancreas and skin of type 2 diabetes mellitus patients [114,115]. It was also identified among 100 genes modified after

intermittent energy restriction (IER) (compared to baseline before IER) in a group of 23 overweight pre-menopausal women. Its expression was decreased in responders to IER together with leptin, adiponectin and lipoprotein lipase [116]. Similarly, in a mouse model of obesity designed to test the anti-obesogenic effect of common bean, *Pcolce2* appeared in the top bean-induced suppressed genes, in both subcutaneous and mesenteric fat, but only in female mice [117].

In cardiovascular diseases, *PCOLCE2* can be co-regulated with genes of the lipid pathway. Thus, in the ECM of bicuspid aortic valves, a congenital valve defect leading to degeneration, several proteins involved in cholesterol metabolism were found to be enriched by mass spectrometry when compared to the degenerated ECM of “normal” tricuspid aortic valves [118]. Among these proteins with higher abundance were the phospholipid transfer protein (PLTP), ApoA4 and the HDL-binding protein vigilin but also PCPE-2. Similarly, in severe aortic stenosis patients, PCPE-2 was found to be up-regulated in fibrotic regions of the aortic valve compared to the non-diseased areas, both at the transcript and protein levels, and was localized to the fibrosa layer of the valve where apolipoproteins were also more abundant in patient samples compared to normal controls [28]. Disease progression was associated with an overrepresentation of lipid-related pathways such as LDL-mediated lipid transport and lipoprotein metabolism.

Finally, genetic associations also revealed possible links between PCPE-2 and fatty acids or lipoproteins. First, a strong genetic link

<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Schematic of the various options to explain the role of PCPE-2 in lipid metabolism. (A) Pro-ApoA1 maturation by BMP-1 leads to the formation of HDL particles, with normal size and lipid content, and PCPE-2 was suggested to enhance this process [52,57]. The final step of HDL biogenesis is the esterification of cholesterol ("C") into cholesteryl ester ("CE"), to yield mature spherical HDL particles with a CE-rich core. This mechanism is controversial as the diminution of pro-ApoA1 processing in the absence of PCPE-2 was not confirmed by another group [122] and independent results suggest that PCPE-2 could also inhibit pro-ApoA1 processing by BMP-1 [19]. (B) PCPE-2 regulates HDL cholesteryl ester cellular uptake through the control of the amount, localization and/or conformation of the SR-BI receptor at the plasma membrane [30,122,123]. This mechanism is presently the most well supported by experimental results and does not involve BMP-1. (C) PCPE-2 could potentiate LDL uptake by LDLR through the inhibition of BMP-1-dependent LDLR cleavage [19]. The biological relevance of this mechanism has not been confirmed in vivo. Created in BioRender [124].</figcaption></figure>
between single nucleotide polymorphisms (SNPs) in *PCOLCE2* locus and arachidonic acid levels in red blood cells was evidenced using genome-wide association studies (GWAS) on >2300 individuals [119]. Arachidonic acid is a fatty acid that is transported by plasma lipoproteins and can be exchanged with the membrane phospholipids of red blood cells or other cells. This genetic association was found to be significant before or after adjustment for dietary covariates [120]. In addition, genetic associations between *PCOLCE2* SNPs and HDL cholesterol levels were also evidenced in three different cohorts [57,121], yet they were not statistically significant after correction for multiple testing. HDL is considered as an anti-atherogenic lipoprotein due to its role in reverse cholesterol transport to the liver, but it can also drive cholesterol uptake and efflux in other cell types (e.g. adipocytes). This finding suggested a weak genetic effect of *PCOLCE2* variants on HDL cholesterol levels and prompted several studies to validate the functional relationship between these two potential partners.

### Regulation of HDL biogenesis via Apoa1

Concomitantly with the discovery of the genetic association between *PCOLCE2* and HDL cholesterol levels, it was demonstrated that BMP-1 is the enzyme responsible for the maturation of pro-ApoA1 into ApoA1, the main protein constituent of HDL [109]. The removal of six residues at the N-terminus of pro-ApoA1 drives the biogenesis of HDL, probably by allowing a conformational change in the apolipoprotein to promote its lipidation. The logical hypothesis that PCPE-2, as a BMP-1 regulator, could modulate this proteolytic maturation was then tested by Zhu *et al.* [57]. Based on *in vitro* experiments made with the medium of CHO cells enriched in PCPE-2, they provided evidence that PCPE-2 can actually enhance BMP-1 maturation of pro-ApoA1 around 2-fold and form a ternary complex with the two proteins, as shown by SPR. However, the mechanism by which PCPE-2 bound to pro-ApoA1 and enhanced its maturation was unclear since SPR experiments gave inconsistent results depending on which protein was immobilized or injected first.

In another study by the same group, the plasma lipid profile of *Pcolce2*-deficient mice was investigated. Interestingly, increased amounts of HDL cholesterol (HDL-C) particles and ApoA1 in plasma were observed when deficient mice were compared to WT mice [52]. This was also true when the mice were fed a high-fat high-cholesterol diet except that the increase in HDL-C was significant only in female mice. The HDL particles were also found to be larger, a finding which could be explained by a defect in pro-ApoA1 maturation or by an increased content in lipids. The first explanation (Fig. 4a) was preferred as a slight increase in the pro-ApoA1/ApoA1 ratio was observed in the absence of PCPE-2 even though it could not be precisely quantified. This study also provided preliminary data suggesting that the HDL particles found in the serum of *Pcolce2*-deficient mice had a reduced capacity to promote the influx and efflux of cholesterol in macrophages [52].

These results were revisited in 2015 by Pollard et al. who confirmed the increased levels of ApoA1 and the enlarged HDL particles in mice lacking PCPE-2 [122]. However, they found that both WT and *Pcolce2*-deficient mice displayed the same low level of pro-ApoA1 in their plasma (around 4 %) and that HDL enlargement and dysfunction in the absence of PCPE-2 was unlikely to be due to the modification of pro-ApoA1 processing. Instead, they proposed and tested another mechanism, based on the regulation of SR-BI, that will be described in the next section. The other finding that challenges the possible enhancement of BMP-1-dependent pro-ApoA1 processing by PCPE-2 is the recent discovery that the latter is a potent inhibitor of BMP-1 for non-collagenous substrates [19], as discussed above. Therefore, the question of the effect of PCPE-2 on ApoA1 maturation by BMP-1 (enhancement/inhibition) and of the relevance of the mechanism described in Fig. 4a remains open.

### Regulation of SR-BI-mediated uptake of HDL

Although Pollard et al. did not confirm the role of PCPE-2 in ApoA1 processing, they provided additional evidence that PCPE-2 is a major

player in lipid metabolism. Indeed, when the *Pcolce2*-deficient mice were crossed with *Ldlr*-null mice and fed an atherogenic diet, they displayed increased lipid accumulation and macrophage infiltration in the aortic root, despite their elevated HDL-C levels [122], suggesting that PCPE-2 is atheroprotective. However, this study also showed that HDL particles were not dysfunctional *per se* but, rather, that they had a slower catabolism causing their accumulation and enlargement in the circulation. HDL catabolism is largely dependent on SR-BI, a cell receptor whose primary role is to mediate the selective uptake of HDL particles loaded with cholesterol into the liver [125,126]. It is also expressed in adipocytes and macrophages where it mediates the bidirectional flux of cholesterol between cells and HDL. Notably, a strong increase in SR-BI levels in mouse liver, adipose tissue and adipocytes lacking PCPE-2 was reported but this compensation was paradoxically associated with a diminution of their capacity for selective HDL uptake [30,122]. When placed in the same atherogenic conditions as above, *Pcolce2*-deficient mice presented with hypertrophic adipocytes and an altered lipid balance between plasma and adipose tissue [30]. The observed dysfunctional uptake could be reversed in adipocytes by the pre-treatment with recombinant PCPE-2, suggesting a stable interaction between SR-BI and PCPE-2 [30]. Xu et al. therefore proposed that PCPE-2 has the capacity to regulate SR-BI localization or multimerization at the plasma membrane [30], thereby directly affecting cholesterol influx and, possibly, its efflux [30,52] (Fig. 4b). All these results suggest an important role of PCPE-2 in the regulation of cholesterol level in plasma, liver and adipose tissue, mainly driven by the SR-BI receptor. The details of the SR-BI/PCPE-2 interaction and its consequence for SR-BI function remain to be established.

### Regulation of LDL-lipid trafficking

A third mechanism by which PCPE-2 could regulate lipid metabolism is extrapolated from recently published *in vitro* studies [19]. It was actually shown that PCPE-2 can completely block BMP-1-dependent processing of human LDLR, a receptor that mediates the endocytosis of LDL particles. This was shown both in an *in vitro* cleavage assay with recombinant BMP-1, PCPE-2 and LDLR ectodomain and in a cellular model of PCPE-2 overexpression. It was already known that BMP-1 cleaves LDLR (Table 2) within its N-terminal ligand binding domain and significantly reduces its ability to bind LDL [110,127,128]. The inhibition of BMP-1 by PCPE-2 could thus limit LDLR processing and promote LDL elimination by cells, thereby decreasing the plasma levels of atherogenic LDL particles (Fig. 4c). However, this mechanism remains putative and could not be confirmed *in vivo* because the LDLR cleavage site is not conserved in mice [110]. Therefore, the fact that *Pcolce2*-deficient mice do not have modified plasma LDL levels compared to WT mice [52,57] does not allow to draw any conclusion about the possible involvement of PCPE-2 in the regulation of LDL catabolism in humans. Appropriate models need to be established to investigate this possibility.

### Role of PCPE-2 in innate immunity and inflammation

The first hints that PCPE-2 has implications in inflammatory diseases were provided by studies on pathological conditions such as severe influenza [129], sepsis and SIRS (severe inflammatory response syndrome) [130] or pancreatitis [131]. In the first example, *PCOLCE2* was up-regulated in patients with severe influenza when compared to patients with non-severe influenza and appeared as one of the two most robust differentially expressed genes to discriminate the two groups [129]. *PCOLCE2* was also up-regulated in sepsis and to a lower extent in non-infectious SIRS where it was identified as a hub gene [130]. In mouse models of chronic pancreatitis, a disease characterized by chronic inflammation and progressive fibrosis, *PCOLCE2* was increased both between the pancreatitis (induced by repetitive injections of cerulein) and control groups and between the more severely (C57BL/6 J) and less severely (C57BL/6NHsd) affected mouse strains [131]. Finally, in secondary hyperparathyroidism, which is a frequent complication of
chronic kidney disease involving inflammation, *PCOLCE2* was also correlated with immune cell infiltration and proposed as a potential biomarker of disease progression [132].

A first but still hypothetical mechanistic link between *PCOLCE2* and inflammation could be through arachidonic acid. As indicated above, SNPs associated with the *PCOLCE2* locus contribute to explain the heritable component of circulating arachidonic acid levels [119,120]. This fatty acid is also the precursor of several potent inflammatory mediators such as prostaglandins and leukotrienes, linking nutrient metabolism with immunity and inflammation. Inhibitors of the cyclooxygenase that converts arachidonic acid into prostaglandins and thromboxane (e.g. aspirin, ibuprofen) are widely used to control inflammation and were found to influence macrophage polarization [133]. Arachidonic acid can also directly stimulate neutrophil activation, for example by triggering an oxidative burst, a major response in anti-microbial defense [134]. Whether PCPE-2 could impact inflammation through the control of arachidonic acid levels would thus be interesting to establish. However, several additional lines of evidence suggest that it plays a direct role in the control of immune cell activity.

While the possible link between the embryonic expression of *Pcolce2* in the thymus and a role in the establishment of adaptive immunity was ruled out by the work of Heinzel and Bleul [22], a number of findings actually indicate that this gene product is important for innate immunity. The preliminary demonstration of this was provided in 2015 by Jiang et al., who observed that the expression level of *PCOLCE2* could reveal disease-specific regulations in neutrophil populations [135]. *PCOLCE2* was indeed found to be one of the most substantially modified genes in neutrophils isolated from the blood of children with cystic fibrosis when compared to children with juvenile idiopathic arthritis. Although the exact function of PCPE-2 in neutrophils was not clear at that time, some interesting clues came a few years later when Yoon et al. demonstrated that the PCPE-2 protein secreted by tonsil mesenchymal stem cells (TMSCs) has a significant influence on neutrophils (in this case promyelocytic HL-60 human cells differentiated into neutrophils *in vitro*) [136]. In the TMSC subset which secrete the highest amounts of PCPE-2, a doubling of the production of reactive oxygen species (ROS) by neutrophils was measured upon PMA (phorbol 12-myristate 13-acetate) stimulation while there was no significant effect with TMSCs secreting low PCPE-2 concentrations in the same conditions. A similar response was obtained when recombinant PCPE-2 was added to neutrophil cultures, in the absence of TMSCs, with a clear increase in ROS production starting at PCPE-2 concentrations as low as 2 ng/ml. Finally, the siRNA-mediated knockdown of *PCOLCE2* expression in TMSCs significantly diminished the enhancing effect on neutrophil ROS production, clearly implicating the PCPE-2 protein in the stimulation of the oxidative burst. Mechanistically, PCPE-2 was found to increase the expression of NADPH oxidases (*NOX*) 3–5 and of dual oxidase (*DUOX*) 1 which are not the preferred NADPH oxidases in neutrophils but could still participate in superoxide generation [136]. Interestingly, PCPE-2 might also elicit other neutrophil responses as it was shown to induce peptidyl arginase deiminase 4 (*PADI4*) and Rac family small GTPase 2 (*RAC2*) which are involved in the formation of neutrophil extracellular traps (NETs) and in phagocytosis respectively. Together, these results clearly point to PCPE-2 having direct immuno-modulatory functions in neutrophils even if it still needs to be established whether PCPE-2 only acts in a paracrine manner or could also work as an autocrine factor synthesized by the neutrophils themselves.

Important results regarding *PCOLCE2* expression were also obtained in macrophages from different origins. In human CD14+ blood monocytes differentiated into macrophages by M-CSF (macrophage colony stimulating factor) treatment, *PCOLCE2* was found to be one of the most highly expressed core matrisome genes (as defined by Naba et al. [137]) [138]. *BMP1* was also expressed but at a lower intermediate level. In the same study, *PCOLCE2* was also one the most downregulated genes upon IFNγ (interferon γ) treatment suggesting that it might be repressed in pro-inflammatory (M1 polarization) macrophages [138]. In two

zebrafish models of heart injury (heart resection and cryoinjury), *Pcolce2b* was found to be specifically increased in macrophages at day 5 post-injury when pro-regeneration/pro-resolution gene signatures were maximum [139]. Similarly, *Pcolce2* dysregulation in macrophages peaked at day 4 in mice with myocardial infarction, although in the opposite direction, and this dysregulation was specific to adult mice compared to neonates which are known to regenerate better [139]. Overall, it seems that PCPE-2 expression in macrophages is tightly associated with their polarization status, leading to a higher expression in alternatively-activated macrophages (M2 polarization), with a potential link with their pro-regenerative capacity.

The link between *PCOLCE2* and macrophages was also nicely highlighted in the context of ovarian carcinoma when tumor-associated macrophages (TAMs) isolated from patient ascites were compared to normal resident peritoneal macrophages (pMPHs) and to monocyte-derived macrophages (MDMs). Strikingly, *PCOLCE2* was the top-ranked gene regarding TAM/pMPH ratios and the third for TAM/MDM ratios [140], suggesting a strong induction in the tumor micro-environment. In contrast, *PCOLCE2* was not found or very lowly expressed in tumor cells while *BMP1* was expressed in both cell types, emphasizing that *BMP1* has a broader distribution than *PCOLCE2*. However, the two gene products are secreted proteins which can meet in the extracellular environment even if they are not produced by the same cells. Of high relevance in this context is the fact that PCPE-2 concentration was measured around 100 ng/ml in ascite samples from high-grade serous ovarian carcinoma patients [140], therefore raising various possibilities to predict its activity during tumor progression. Either PCPE-2 could inhibit secreted BMP-1, produced by TAMs or cancer cells, or it could regulate other BMP-1-independent pathways or both. TAMs were shown to share most of the functions of resident macrophages such as phagocytosis, antigen presentation and cytotoxic T cell activation but seem to be skewed to alternative/M2 polarization through the release of immunosuppressive mediators and a lack of response to inflammatory stimuli. Based on these features, TAMs are considered to be mostly pro-tumorigenic but different TAM subgroups can be correlated to more or less favorable outcomes. In this line, *PCOLCE2* in association with CD163 (a marker of M2 activation) and IL6 (a cytokine promoting tumor growth) was used as a biomarker to differentiate TAMs from patients with good or bad prognosis [141]. By RNAseq, the high expression of these markers was found to be associated with genes involved in ECM remodeling, whereas their low expression pointed to IFN signaling as an enriched pathway and a possible therapeutic option to stimulate cytotoxic immune responses. Finally, macrophage infiltration can also accelerate the progression of other cancer types and *PCOLCE2* was recently identified as a macrophage-related dysregulated gene in colorectal cancer patient samples [142]. Together, these results indicate that PCPE-2 is crucial to dictate or mediate the polarization status of macrophages and their adaptation to the microenvironment but much remains to be done to decipher underlying molecular mechanisms.

## Role of PCPE-2 in cancer progression

In the DISEASES database, which integrates information on disease-gene associations mined from literature [143], the highest association score for *PCOLCE2* is with cancer, while for *BMP1*, it is the second best score after Osteogenesis Imperfecta. A nice illustration of this association is given by the finding that *PCOLCE2* is among the top ten down-regulated matrisome genes when tumour samples from 15 different origins (transcriptomic data available from The Cancer Genome Atlas or TCGA dataset) are compared to normal tissues (from the TCGA and GTEx datasets) [45]. Although not as reliable due to small numbers of control samples for certain cancer types, the survey of a larger TCGA dataset including 24 cancer types provided by the UALCAN database [144,145] confirms a strong downregulation trend for *PCOLCE2* in tumours (Fig. 5A). Most of the other downregulated matrisome genes
<figure><img src="image_11.png" /><figcaption>**Fig. 5.** Expression of *PCOLCE2* (A) and *BMP1* (B) in different types of primary tumors (red) in comparison with normal tissues (blue). Transcriptomic data originate from UALCAN (https://ualcan.path.uab.edu/cgi-bin/Pan-cancer.pl?genenam=PCOLCE2 and https://ualcan.path.uab.edu/cgi-bin/Pan-cancer.pl?genenam=BMP1). BLCA, Bladder urothelial carcinoma (n = 19 normal samples, n = 408 primary tumors); BRCA, Breast invasive carcinoma (n = 114 normal samples, n = 1097 primary tumors); CESC, Cervical squamous cell carcinoma (n = 3 normal samples, n = 305 primary tumors); CHOL, Cholangiocarcinoma (n = 9 normal samples, n = 36 primary tumors); COAD, Colon adenocarcinoma (n = 41 normal samples, n = 286 primary tumors); ESCA, Esophageal carcinoma (n = 11 normal samples, n = 184 primary tumors); GBM, Glioblastoma multiforme (n = 5 normal samples, n = 156 primary tumors); HNSC, Head and neck squamous cell carcinoma (n = 44 normal samples, n = 520 primary tumors); KICH, Kidney chromophobe (n = 25 normal samples, n = 67 primary tumors); KIRC, Kidney renal clear cell carcinoma (n = 72 normal samples, n = 533 primary tumors); KIRP, Kidney renal papillary cell carcinoma (n = 32 normal samples, n = 290 primary tumors); LIHC, Liver hepatocellular carcinoma (n = 50 normal samples, n = 371 primary tumors); LUAD, Lung adenocarcinoma (n = 59 normal samples, n = 515 primary tumors); LUSC, Lung squamous cell carcinoma (n = 52 normal samples, n = 503 primary tumors); PAAD, Pancreatic adenocarcinoma (n = 4 normal samples, n = 178 primary tumors); PRAD, Prostate adenocarcinoma (n = 52 normal samples, n = 497 primary tumors); PCPG, Pheochromocytoma and paraganglioma (n = 3 normal samples, n = 179 primary tumors); READ, Rectum adenocarcinoma (n = 10 normal samples, n = 166 primary tumors); SARC, Sarcoma (n = 2 normal samples, n = 260 primary tumors); SKCM, Skin cutaneous melanoma (n = 1 normal samples, n = 104 primary tumors); THCA, Thyroid carcinoma (n = 59 normal samples, n = 505 primary tumors); THYM, Thymoma (n = 2 normal samples, n = 120 primary tumors); STAD, Stomach adenocarcinoma (n = 34 normal samples, n = 415 primary tumors); UCEC, Uterine corpus endometrial carcinoma (n = 35 normal samples, n = 546 primary tumors). \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001; ns=non-significant.</figcaption></figure>

co-expressed with *PCOLCE2* [45] are already known to play a role in tumour progression and/or have also been validated at the protein level, for example *WISP2* [146], *ADAM33* [147], *DPT* [148] and *TNXB* [149], providing high confidence in the relevance of *PCOLCE2* downregulation. It has been suggested that this diminution of expression could be linked to DNA hypermethylation, at least in nasopharyngeal carcinoma [150]. Notable exceptions are liver hepatocellular carcinoma and glioblastoma (Fig. 5a: LIHC, GBM) where *PCOLCE2* seems to be upregulated rather than downregulated. Glioblastoma is one of the rare examples where *PCOLCE2* dysregulation has also been confirmed at the protein level [151] and the inverse trend appears relatively reliable (Table 3).

In contrast to the broad downregulation observed for *PCOLCE2*, *BMP1* appears to be significantly upregulated in more than half of the analyzed cancer types (Fig. 5b). This could mean that, in these tumors with high *BMP1* expression, PCPE-2 activity must be turned off to allow BMP-1 pro-tumorigenic activity. As illustrated in Table 2, BMP-1 can

regulate tumor growth through several mechanisms such as promoting collagen and ECM deposition, contributing to TGF-β activation, modulating angiogenesis and releasing tumor-promoting adipokines. Several studies also describe the direct role of BMP-1 in cancer progression and metastasis and although underlying mechanisms remain elusive, BMP-1 was shown to promote migration, invasion and epithelial-to-mesenchymal transition (EMT) [85,186–190]. EMT is a major mechanism in cancer progression, often triggered by TGF-β [191], by which a polarized epithelial cell turns into a mesenchymal cell with enhanced migratory and invasive capacities. However, BMP-1 was also described as protective against tumor growth and metastasis. Especially, in the context of pancreatic ductal adenocarcinoma (PDAC), the BMP-1 protein specifically secreted by tumor cells (as opposed to stromal cells) was found to exert beneficial functions through the production of more adequately-matured collagens [192].

It is not clear at this point if the role of PCPE-2 in cancer is only to
<table><caption>**Table 3**

PCOLCE2 regulation and prognostic value in cancer.</caption><thead><tr><th>Cancer type</th><th>Up/down regulation in cancer</th><th>Prognostic gene linked to survival Y/ N</th><th>Comments</th><th>References</th></tr></thead><tbody><tr><td>Bladder cancer</td><td>Down</td><td>Y</td><td>High expression linked to poor survival, immune infiltration, low response to therapy and aggressiveness</td><td>[45,144, 152–155]</td></tr><tr><td>Breast cancer</td><td>Down</td><td>Y</td><td></td><td>[45,144, 156–158]</td></tr><tr><td>Colorectal cancer</td><td>Down (also in cancer cell lines and in adipocytes close to tumor site)</td><td>Y</td><td>High expression linked to poor survival, immune infiltration, hypoxia, angiogenesis and EMT scores, aggressiveness and high expression of HMGA2</td><td>[45,142,144, 159,160–169]</td></tr><tr><td>Esophageal carcinoma</td><td>Up</td><td>Y</td><td>High expression linked to poor survival and aggressiveness</td><td>[170–172]</td></tr><tr><td>Gastric cancer</td><td>Down</td><td>Y</td><td>High expression linked to poor survival; Possible link to mutation in PCOLCE2 gene</td><td>[45,144, 173–175]</td></tr><tr><td>Glioblastoma</td><td>Up</td><td>Y</td><td>High expression linked to poor survival</td><td>[144,151]</td></tr><tr><td>Head and neck squamous cell carcinoma</td><td>Down</td><td>Y</td><td>High expression linked to poor survival and low response to therapy</td><td>[176]</td></tr><tr><td>Liver hepatocellular carcinoma</td><td>Up</td><td></td><td>High expression linked to poor survival</td><td>[45,144]</td></tr><tr><td>Lung adeno-carcinoma</td><td>Down (also in fibroblasts close to tumor site)</td><td></td><td></td><td>[45,74,144,177]</td></tr><tr><td>Lung squamous cell carcinoma</td><td>Down</td><td></td><td></td><td>[144,178]</td></tr><tr><td>Nasopharyngeal carcinoma</td><td>Down</td><td></td><td>Low expression linked to DNA hypermethylation</td><td>[150]</td></tr><tr><td>Non-small cell lung cancer</td><td>Down</td><td>Y</td><td></td><td>[179,180]</td></tr><tr><td>Ovarian cancer</td><td>Down</td><td></td><td></td><td>[45]</td></tr><tr><td>Ovarian carcinoma</td><td>Up in TAMs versus MDMs and pMPHs</td><td>Y</td><td>Analysis of TAMs, not tissue; High expression linked to aggressiveness</td><td>[140,141]</td></tr><tr><td>Prostate adenocarcinoma</td><td>Down</td><td></td><td></td><td>[45,144]</td></tr><tr><td>Thyroid carcinoma</td><td>Down</td><td>Y</td><td>High expression linked to poor survival and immune infiltration</td><td>[45,144,181, 182–184]</td></tr><tr><td>Uterine corpus endometrial carcinoma</td><td>Down</td><td>Y</td><td>High expression linked to poor survival</td><td>[144,185]</td></tr></tbody></table>

counteract BMP-1 activity. Actually, the study recently published by Zhou et al. [152] suggests the opposite. They proposed a prognostic gene-signature in bladder cancer involving cancer associated-fibroblasts (CAFs), a major cell type of the tumor microenvironment. CAFs are known to secrete different factors such as chemokines, collagens and metalloproteinases [193] and therefore participate in tumorigenesis. The presence of CAFs and associated desmoplasia are among the most prominent features of invasive tumours in bladder cancer [194]. After demonstrating that PCOLCE2 is a CSE (CAFs-Stromal-EMT) -related gene in bladder cancer, the authors used viral transfection to overexpress PCOLCE2 in T24 cells (a bladder cancer cell line) and observed increased cell migration and invasion. This property was associated with a decrease in E-cadherin expression and an increase in vimentin and N-cadherin, which are typical markers of EMT. Similarly, in colon adenocarcinoma cell lines, Yin et al. revealed that the siRNA-mediated invalidation of PCOLCE2 leads to an increase in E-cadherin expression, confirming the potential role of the gene product in EMT [170]. However, colon adenocarcinoma is one of the few examples of cancers for which there is no consensus on the regulation of PCOLCE2 expression (up or down; Table 3). Other hypotheses to explain the role of PCPE-2 in this cancer type are increased resistance to anoikis [159] and abnormal cholesterol metabolism through PCPE-2 involvement in lipid transport (see above). In summary, as both PCPE-2 and BMP-1 overexpression seem to induce similar effects (migration, invasion, EMT), further studies are needed to determine if they act in a concerted manner or independently.

Finally, an increasing number of studies also highlight that PCOLCE2 could be a valuable diagnostic and prognostic marker. It was indeed proposed to predict cancer or cancer stage, survival, metastasis, immune infiltration, immunotherapy benefit or resistance to therapy (Table 3). For example, in 2017, Lim et al. developed a 29-gene ECM-related prognostic indicator in non-small cell lung cancer [179]. A recent

publication also included PCOLCE2 in an EMT-related gene signature of 4 genes for gastric cancer [173]. Although PCOLCE2 was overall downregulated in tumour tissue compared to normal tissue, a high level of expression was correlated with poorer survival outcome. The same observation was reported in several other cancer types (Table 3: bladder, uterine, colorectal, thyroid, head and neck cancers) and is often associated with higher grade and/or metastatic stage [141,153] and poorer response to treatment [153–155,176]. This apparent discrepancy (lower expression in tumour compared to normal tissue but high expression in tumours with poor prognosis) is presently difficult to explain. One possible explanation could be that different cell types are responsible for PCPE-2 production in normal and low/high grade cancer tissues. CAFs and TAMs are possible candidates. While PCOLCE2 expression was found to be decreased >5-fold when comparing CAFs from a pan-cancer dataset with non-CAF fibroblasts [45], it is around 20-fold higher in TAMs from ovarian carcinoma ascites than in control macrophages (Table 3 and previous section). Furthermore, PCPE-2 concentration in ascites can be used to differentiate TAMs from patients with good or bad prognosis [141]. A similar link between PCOLCE2 and macrophage infiltration also seems to apply to colorectal [142] and thyroid [181] cancers. Overall, PCOLCE2 expression seems highly dysregulated in cancer and linked to prognosis but elucidating the precise role of this gene in disease progression and survival deserves more investigation.

## Conclusion and future directions

Several challenges have hampered the detailed characterization of PCPE-2 until now. First, its high structural similarity with PCPE-1 has led to the misleading belief that it would behave exactly the same and did not deserve extensive analysis. Also, its rather low tissue and cell expression combined with the questionable specificity of anti-PCPE-2 antibodies makes its immuno-detection particularly tricky. However,
non-targeted omics approaches have shown that it is present in a relatively broad range of tissues which will probably continue to expand in the future. Notably, a distinctive feature of the PCPE-2 protein seems to be its preferential localization to specific organ structures (e.g. trabecular meshwork in the eye, cochlea in the ear, fibrosa layer of the aortic valve, anterior lobe of the pituitary gland, reticular dermis in skin) or cell types (e.g. podocytes in kidney, trophoblastic cells in placenta, mesenchymal stem cells in tonsils, fibro-adipogenic precursors in skeletal muscle). This suggests specific functions and subtle regulatory mechanisms at the organ level. Other challenges for *in vitro* studies are the relative lack of stability of PCPE-2 in standard buffer conditions and its propensity to attach to the cell layer in cell culture assays. Efficient recombinant systems in mammalian cells have now been established for the full-length human protein but expression of mutant proteins often requires more complicated strategies.

The main objective for future research should be to decipher the precise functions of PCPE-2 in pathophysiological conditions and to link them with mechanisms of action at the molecular level. As emphasized in this review, the canonical role of a procollagen C-proteinase enhancer defined for PCPE-1 is not appropriate to describe PCPE-2 activities. The recent discovery that PCPE-2 also works as a specific inhibitor of BTPs opens new unexplored avenues to better understand its functions, yet the *in vivo* relevance of this finding remains to be demonstrated. In addition, some pathologies such as atherosclerosis, inflammatory diseases and cancer seem to involve PCPE-2 but its exact role still has to be described. Inflammation and lipid metabolism appear to be relevant biological settings to gain additional insights and many more certainly remain to be discovered. PCPE-2 has not yet revealed its full potential, either as a research topic or as a therapeutic target.

## Declaration of competing interest

The authors declare that they have no competing interests.

## Data availability

Data were extracted from public databases or results published elsewhere.

## Acknowledgements

We thank Audrey Ziverec and Violaine Sée (LBTI, Lyon) for the critical reading of the manuscript and Juliette Hof for the help with the preparation of the artwork.

## Funding

This work was supported by CNRS, Université Claude Bernard Lyon I and Agence Nationale de la Recherche [grant number ANR-21-CE11-0020-01].

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