Procollagen C-proteinase Enhancer Research Articles

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-β) enhanced procollagen synthesis by 45 ± 3% (P < 0.001), and 38 ± 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 ± 8% (P < 0.01) and 72% ± 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-β-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.

Structural Organization and Expression Patterns of the Human and Mouse Genes for the Type I Procollagen COOH-Terminal Proteinase Enhancer Protein

The procollagen C-proteinase enhancer (PCPE) is a glycoprotein that potentiates enzymatic cleavage of the type I procollagen C-propeptide by bone morphogenetic protein-1 (BMP-1). The human PCPE gene (PCOLCE) was previously mapped to 7q22, an area frequently disrupted in uterine leiomyomata, while disruption of the rat PCPE gene leads to anchorage-independent growth and loss of contact inhibition in rat fibroblasts. Here we describe the entire intron/exon organizations ofPCOLCEand the mouse PCPE gene (Pcolce) and analyze expression ofPCOLCERNA in various human adult and fetal tissues and ofPcolceRNA at various stages of mouse development.PCOLCEandPcolceare shown to be small genes 6.0 and 6.5 kb, respectively, with a conserved intron/exon structure comprising 9 exons. A notable difference between the two genes derives from insertion of multipleAlusequences immediately upstream and downstream and withinPCOLCE.Temporal expression of PCPE mRNA is shown to differ from that of BMP-1 and type I procollagen during mouse development, consistent with possible additional functions for PCPE beyond enhancement of C-proteinase activity. Consistent with a possible role in leiomyomata,PCOLCEis shown to be expressed at relatively high levels in uterus.

The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.

Using homology search, structure prediction, and structural characterization methods we show that the C-terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled-related proteins, and (4) type I procollagen C-proteinase enhancer proteins (PCOLCEs) are homologous with the N-terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin-type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin-type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis.

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression.

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression. (Hepatology 1997 Sep;26(3):611-7)

Transforming growth factor-beta regulation of bone morphogenetic protein-1/procollagen C-proteinase and related proteins in fibrogenic cells and keratinocytes.

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

The CUB domains of procollagen C-proteinase enhancer control collagen assembly solely by their effect on procollagen C-proteinase/bone morphogenetic protein-1.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

Procollagen C-proteinase enhancer is localised to dense connective tissue

Procollagen C-proteinase enhancer is localised to dense connective tissue

Procollagen type I C-proteinase enhancer is a naturally occurring connective tissue glycoprotein

Using antibodies to the procollagen C-proteinase enhancer of mouse fibroblast culture medium, we have screened by immunoblotting extracts of several post natal mouse and rat tissues for the presence of the enhancer antigen. All rodent connective tissues were relatively rich in enhancer; lower amounts were found in skeletal muscle and heart and essentially no enhancer was detected in kidney, liver or brain. The amounts of enhancer in mouse tendon and calvaria extracts were age related, with highest amounts in 11 and 19 d tendons and in 1 d calvaria-the times of rapid growth of these organs. The results suggest that procollagen C-proteinase enhancer is a specific connective tissue glycoprotein that is likely to regulate procollagen processing in vivo.

Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein.

The enzyme procollagen C-proteinase removes the carboxy-terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl-Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy-terminal propeptide of type I procollagen (PP-Sepharose). This procedure yielded a practically homogeneous, 18,500-fold-purified enzyme preparation and the molecular mass of the purified C-proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl-Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55-kDa protein which was further purified by PP-Sepharose chromatography and identified as an additional form of the 36-kDa and 34-kDa procollagen C-proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6,267-277]. It enhanced the C-proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross-reacted immunologically with the 36-kDa as well as the 34-kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP-Sepharose chromatography, a large proportion of the 55-kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55-kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl-Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C-proteinase from other sources as well.