Kazuhiko Takahara

Bone morphogenetic protein-1 : the type I procollagen C-proteinase

Bone morphogenetic proteins (BMPs) are bone-derived factors capable of inducing ectopic bone formation. Unlike other BMPs, BMP-1 is not like transforming growth factor-β (TGF-β), but it is the prototype of a family of putative proteases implicated in pattern formation during development in diverse organisms. Although some members of this group, such as Drosophila tolloid (TLD), are postulated to activate TGF-β-like proteins, actual substrates are unknown. Procollagen C-proteinase (PCP) cleaves the COOH-propeptides of procollagens I, II, and III to yield the major fibrous components of vertebrate extracellular matrix. Here it is shown that BMP-1 and PCP are identical. This demonstration of enzymatic activity for a BMP-1/TLD-like protein links an enzyme involved in matrix deposition to genes involved in pattern formation.

Bone morphogenetic protein 1 is an extracellular processing enzyme of the laminin 5 gamma 2 chain

Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (α3β3γ2) within the α3 and γ2 chains (1). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first α3 chain cleavage (200-l65 kDa α3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa α3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The γ chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the γ2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (2). Recombinant BMP-1 cleaves γ2 in vitro,both within intact laminin 5 and at the predicted site of a recombinant γ2 short arm. α3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin α3 and γ2 chains to be substrates for BMP-1 in vitro. We speculate that γ2 cleavage is required for formation of the laminin 5–6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.

Transforming Growth Factor-β Regulation of Bone Morphogenetic Protein-1/Procollagen C-proteinase and Related Proteins in Fibrogenic Cells and Keratinocytes

Transforming growth factor-β1 (TGF-β1) 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-β1 and that secreted BMP-1, induced by TGF-β1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-β1 elevated levels of BMP-1 mRNA ∼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-β1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-β1 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-β1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

Order of Intron Removal Influences Multiple Splice Outcomes, Including a Two-Exon Skip, in a COL5A1 Acceptor-Site Mutation That Results in Abnormal Pro-α1(V) N-Propeptides and Ehlers-Danlos Syndrome Type I

Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

PCOLCE deletion and expression analyses in uterine leiomyomata

Uterine leiomyomata (UL) are benign tumors affecting many women of reproductive age. Cytogenetic studies have indicated that a significant percentage of leiomyomata have chromosomal rearrangements, including those involving the long arm of chromosome 7. Several candidate genes that map to chromosome 7 have been studied for possible roles in the pathogenesis of these tumors. PCOLCE, a gene whose product is involved in the cleavage of type I procollagen C-propeptide, has been mapped to the critical interval on chromosome 7, band q22. Here we evaluate by reverse-transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization the expression and deletion status of PCOLCE in a series of karyotyped uterine leiomyomata.