Ucma, a Novel Secreted Cartilage-specific Protein with Implications in Osteogenesis

Abstract

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.Elucidation of molecular mechanisms underlying chondrocyte differentiation is not only important for our understanding of skeletal development, but also of particular interest for our knowledge on the behavior of chondrocytes following articular cartilage damage during cartilage repair and treatment of degenerative cartilage diseases. Initial steps of chondrogenesis, i.e. the formation of a cartilage blastema from limb bud mesenchymal cells, include cell condensation and onset of chondrocyte differentiation marked by the expression of cartilage-specific matrix proteins such as aggrecan, collagen II, IX, and XI and others (1, 2). These events are regulated by the orchestrated action of several growth factors including BMPs, Wnt factors, FGFs, and the transcription factors Sox5,6, and 9 (3,4). Further steps of chondrocyte growth, maturation, and replacement by bone in the growth plate of long bones, ribs, and vertebrae during endochondral ossification can be defined by the stepwise onset or decline of differentially expressed genes: collagen II and Sox9 for resting and proliferating, FGFR3 for proliferating and prehypertrophic, Ihh and PTHrP receptor for prehypertrophic, collagen X for hypertrophic, and Runx2, osteocalcin, and MMP13 for late hypertrophic chondrocytes (...

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification

“…Epiphyseal and hypertrophic cartilage was dissected under the binocular from 5-day-old (P5) Sox9-transgenic and wild-type long bones and digested with trypsin and collagenase as described (Surmann-Schmitt et al, 2008). Chondrocytes were cultured in DMEM/F12 containing 5% FCS for 1 day and total RNA was harvested using an RNeasy Kit (Qiagen).…”

Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis

“…For mRNA expression analyses, RT-PCR and real-time RT-PCR were carried out as previously reported (Schmidl et al, 2006;Surmann-Schmitt et al, 2008). Briefly, total RNA from limb-bud micromass cultures was isolated using the RNeasy Kit (Qiagen) including the optional DNase digestion step.…”

“…Protein immunodetection was carried out as outlined before using anti-His (Cell Signaling Technology, Danvers, MA), anti-HA (Sigma), anti-Wif-1 and anti-b-catenin (H-102 Santa Cruz Biotechnology) antibodies at a dilution of 1:1000 (Schmidl et al, 2006;Surmann-Schmitt et al, 2008).…”

Genetic association study of UCMA/GRP and OPTN genes (PDB6 locus) with Paget's disease of bone

“…This highly conserved protein may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilagebone interface as well as in the early phase of chondrocyte differentiation [14,15]. More recently, the UCMA/GRP protein was suggested to directly influence mineral formation and to play a role in processes involving soft tissue mineralization and abnormal calcification in the vascular system [16].…”