The expression of proprotein convertase PACE4 is highly regulated by Hash-2 in placenta: possible role of placenta-specific basic helix-loop-helix transcription factor, human achaete-scute homologue-2

PACE4 is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which contribute to the activation of transforming growth factor (TGF) beta family proteins. We previously reported that PACE4 is highly expressed in syncytiotrophoblasts of human placenta [Tsuji et al. (2003) BIOCHIM: Biophys. Acta 1645, 95-104]. In this study, the regulatory mechanism for PACE4 expression in placenta was analyzed using a human placental choriocarcinoma cell line, BeWo cells. Promoter analysis indicated that an E-box cluster (E4-E9) in the 5'-flanking region of the PACE4 gene acts as a negative regulatory element. The binding of human achaete-scute homologue 2 (Hash-2) to the E-box cluster was shown by gel mobility-shift assay. The overexpression of Hash-2 caused a marked decrease in PACE4 gene expression. When BeWo cells were grown under low oxygen (2%) conditions, the expression of Hash-2 decreased, while that of PACE4 increased. In both cases, other SPCs, such as furin, PC5/6, and PC7/8, were not affected. Further, PACE4 expression was found to be developmentally regulated in rat placenta. By in situ hybridization, Mash-2 (mammalian achaete-scute homologue 2) mRNA was found to be expressed in the spongiotrophoblast layer where PACE4 was not expressed. In contrast, the PACE4 mRNA was expressed mainly in the labyrinthine layer where Mash-2 was not detected. These results suggest that PACE4 expression is down-regulated by Hash-2/Mash-2 in both human and rat placenta and that many bioactive proteins might be regulated by PACE4 activity.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix. Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-beta (TGFbeta)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFbeta-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFbeta-related factors' biological activity.     

Secretory proprotein convertases PACE4 and PC6A are heparin-binding proteins which are localized in the extracellular matrix: Potential role of PACE4 in the activation of proproteins in the extracellular matrix

PACE4, PC6 and furin are potent subtilisin-like proprotein convertases (SPCs) which are responsible for the activation of transforming growth factor-β (TGFβ)-related factors such as bone morphogenetic proteins. Heparan sulfate proteoglycan within the extracellular matrix (ECM) is known to regulate the biological activity of various differentiation factors including TGFβ-related molecules. PACE4 binds tightly to heparin and its heparin-binding region was found to be a cationic stretch of amino acids between residues 743 and 760. Furthermore, PACE4 was detected in the extracellular material fraction of the HEK293 cells, defined as the material remaining on the culture plate following the removal of the cells from the plate. PACE4 bound to the extracellular fraction was selectively dislodged by heparin into the culture medium. Heparin has no inhibitory activity against PACE4. Similarly, PC6A is also able to bind to heparin, whereas soluble furin does not. In human placenta, PACE4 is mainly present in syncytiotrophoblasts and can be released by heparin. These results suggest that PACE4 and PC6 are unique SPC family proteases that anchor heparan sulfate proteoglycans at the ECM. The interaction between PACE4 and heparan sulfate proteoglycans might play an important role in the delicate spatiotemporal regulation of TGFβ-related factors' biological activity.     

Pleiotrophin expression during odontogenesis

Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

A proteomic approach reveals transient association of reticulocalbin-3, a novel member of the CREC family, with the precursor of subtilisin-like proprotein convertase, PACE4

SPCs (subtilisin-like proprotein convertases) are a family of seven structurally related serine endoproteases that are involved in the proteolytic activation of proproteins. In an effort to examine the substrate protein for PACE4 (paired basic amino-acid-cleaving enzyme-4), an SPC, a potent protein inhibitor of PACE4, an alpha1-antitrypsin RVRR (Arg-Val-Arg-Arg) variant, was expressed in GH4C1 cells. Ectopic expression of the RVRR variant caused accumulation of the 48 kDa protein in cells. Sequence analysis indicates that the 48 kDa protein is a putative Ca2+-binding protein, RCN-3 (reticulocalbin-3), which had previously been predicted by bioinformatic analysis of cDNA from the human hypothalamus. RCN-3 is a member of the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of multiple EF-hand Ca2+-binding proteins localized to the secretory pathway. The most interesting feature of the RCN-3 sequence is the presence of five Arg-Xaa-Xaa-Arg motifs, which represents the target sequence of SPCs. Biosynthetic studies showed that RCN-3 is transiently associated with proPACE4, but not with mature PACE4. Inhibition of PACE4 maturation by a Ca2+ ionophore resulted in accumulation of the proPACE4-RCN-3 complex in cells. Furthermore, autoactivation and secretion of PACE4 was increased upon co-expression with RCN-3. Our findings suggest that selective and transient association of RCN-3 with the precursor of PACE4 plays an important role in the biosynthesis of PACE4.     

Expression of clock proteins in developing tooth

Morphological and functional changes during ameloblast and odontoblast differentiation suggest that enamel and dentin formation is under circadian control. Circadian rhythms are endogenous self-sustained oscillations with periods of 24h that control diverse physiological and metabolic processes. Mammalian clock genes play a key role in synchronizing circadian functions in many organs. However, close to nothing is known on clock genes expression during tooth development. In this work, we investigated the expression of four clock genes during tooth development. Our results showed that circadian clock genes Bmal1, clock, per1, and per2 mRNAs were detected in teeth by RT-PCR. Immunohistochemistry showed that clock protein expression was first detected in teeth at the bell stage (E17), being expressed in EOE and dental papilla cells. At post-natal day four (PN4), all four clock proteins continued to be expressed in teeth but with different intensities, being strongly expressed within the nucleus of ameloblasts and odontoblasts and down-regulated in dental pulp cells. Interestingly, at PN21 incisor, expression of clock proteins was down-regulated in odontoblasts of the crown-analogue side but expression was persisting in root-analogue side odontoblasts. In contrast, both crown and root odontoblasts were strongly stained for all four clock proteins in first molars at PN21. Within the periodontal ligament (PDL) space, epithelial rests of Malassez (ERM) showed the strongest expression among other PDL cells. Our data suggests that clock genes might be involved in the regulation of ameloblast and odontoblast functions, such as enamel and dentin protein secretion and matrix mineralization.     

Follistatin regulates enamel patterning in mouse incisors by asymmetrically inhibiting BMP signaling and ameloblast differentiation

Rodent incisors are covered by enamel only on their labial side. This asymmetric distribution of enamel is instrumental to making the cutting edge sharp. Enamel matrix is secreted by ameloblasts derived from dental epithelium. Here we show that overexpression of follistatin in the dental epithelium inhibits ameloblast differentiation in transgenic mouse incisors, whereas in follistatin knockout mice, ameloblasts differentiate ectopically on the lingual enamel-free surface. Consistent with this, in wild-type mice, follistatin was continuously expressed in the lingual dental epithelium but downregulated in the labial epithelium. Experiments on cultured tooth explants indicated that follistatin inhibits the ameloblast-inducing activity of BMP4 from the underlying mesenchymal odontoblasts and that follistatin expression is induced by activin from the surrounding dental follicle. Hence, ameloblast differentiation is regulated by antagonistic actions of BMP4 and activin A from two mesenchymal cell layers flanking the dental epithelium, and asymmetrically expressed follistatin regulates the labial-lingual patterning of enamel formation.     

Inactivation of proprotein convertase, PACE4, by alpha1-antitrypsin Portland (alpha1-PDX), a blocker of proteolytic activation of bone morphogenetic protein during embryogenesis: evidence that PACE4 is able to form an SDS-stable acyl intermediate with alpha1-PDX.

PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui et al. reported that the ectopic expression of alpha1-antitrypsin variant Portland (alpha1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y. et al. (1998) EMBO J. 17, 4735-4743]. TGFbeta-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an alpha1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to alpha1-PDX, have been reported for PACE4. In this study, we examined whether alpha1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that alpha1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4 in vivo and in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An alpha1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease(s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease(s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca(2+)-dependent protease with an optimal Ca(2+) requirement of 2 mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by alpha1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by alpha1-PDX causes abnormal embryogenic development.     

Differential expression of decorin and biglycan genes during mouse tooth development.

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.     

Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.     

Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.