Cthrc1 is a positive regulator of osteoblastic bone formation

Background

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.

Methodology/Principal Findings

We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.

Conclusions/Significance

Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis.     

Cthrc1, a Novel Circulating Hormone Regulating Metabolism

Background

We discovered the gene Collagen Triple Helix Repeat Containing 1 (Cthrc1) and reported its developmental expression and induction in adventitial cells of injured arteries and dermal cells of skin wounds. The role of Cthrc1 in normal adult tissues has not yet been determined.

Methodology/Principal Findings

We generated mutant mice with a novel Cthrc1 null allele by homologues recombination. Cthrc1 null mice appeared developmentally normal. On the C57BL/6J background, livers from Cthrc1 null mice accumulated vast quantities of lipid, leading to extensive macrovesicular steatosis. Glycogen levels in skeletal muscle and liver of Cthrc1 null mice on the 129S6/SvEv background were significantly increased. However, Cthrc1 expression is not detectable in these tissues in wild-type mice, suggesting that the lipid and glycogen storage phenotype may be a secondary effect due to loss of Cthrc1 production at a distant site. To investigate potential hormonal functions of Cthrc1, tissues from adult mice and pigs were examined for Cthrc1 expression by immunohistochemistry with monoclonal anti-Cthrc1 antibodies. In pigs, Cthrc1 was detected around chromophobe cells of the anterior pituitary, and storage of Cthrc1 was observed in colloid-filled follicles and the pituitary cleft. Pituitary follicles have been observed in numerous vertebrates including humans but none of the known pituitary hormones have hitherto been detected in them. In C57BL/6J mice, however, Cthrc1 was predominantly expressed in the paraventricular and supraoptic nucleus of the hypothalamus but not in the posterior pituitary. In human plasma, we detected Cthrc1 in pg/ml quantities and studies with ¹²⁵I-labeled Cthrc1 revealed a half-life of 2.5 hours in circulation. The highest level of Cthrc1 binding was observed in the liver.

Conclusions

Cthrc1 has characteristics of a circulating hormone generated from the anterior pituitary, hypothalamus and bone. Hormonal functions of Cthrc1 include regulation of lipid storage and cellular glycogen levels with potentially broad implications for cell metabolism and physiology.     

Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes

During endochondral ossification, type I collagen is synthesized by osteoblasts together with some hypertrophic chondrocytes. Type I collagen has also been reported to be progressively synthesized in degenerative joints. Because Matrix Metalloproteinase-13 (MMP-13) plays an active role in remodeling cartilage in fetal development and osteoarthritic cartilage, we investigated whether type I collagen could activate MMP-13 expression in chondrocytes. We used a well-established chondrocytic cell line (MC615) and we found that MMP-13 expression was induced in MC615 cells cultured in type I collagen gel. We also found that alpha1beta1 integrin, a major collagen receptor, was expressed by MC615 cells and we further assessed the role of alpha1beta1 integrin in conducting MMP-13 expression. Induction of MMP-13 expression by collagen was potently and synergistically inhibited by blocking antibodies against alpha1 and beta1 integrin subunits, indicating that alpha1beta1 integrin mediates the MMP-13-inducing cellular signal generated by three-dimensional type I collagen. We also determined that activities of tyrosine kinase and ERK and JNK MAP kinases were required for this collagen-induced MMP-13 expression. Interestingly, bone morphogenetic protein (BMP)-2 opposed this induction, an effect that may be related to a role of BMP-2 in the maintenance of cartilage matrix.     

Type X collagen expression in osteoarthritic and rheumatoid articular cartilage

Type X collagen is a short chain, non-fibrilforming collagen synthesized primarily by hypertrophic chondrocytes in the growth plate of fetal cartilage. Previously, we have also identified type X collagen in the extracellular matrix of fibrillated, osteoarthritic but not in normal articular cartilage using biochemical and immunohistochemical techniques (von der Mark et al. 1992 a). Here we compare the expression of type X with types I and II collagen in normal and degenerate human articular cartilage by in situ hybridization. Signals for cytoplasmic α1(X) collagen mRNA were not detectable in sections of healthy adult articular cartilage, but few specimens of osteoarthritic articular cartilage showed moderate expression of type X collagen in deep zones, but not in the upper fibrillated zone where type X collagen was detected by immunofluorescence. This apparent discrepancy may be explained by the relatively short phases of type X collagen gene activity in osteoarthritis and the short mRNA half-life compared with the longer half-life of the type X collagen protein. At sites of newly formed osteophytic and repair cartilage, α1(X) mRNA was strongly expressed in hypertrophic cells, marking the areas of endochondral bone formation. As in hypertrophic chondrocytes in the proliferative zone of fetal cartilage, type X collagen expression was also associated with strong type II collagen expression.     

Ectopic osteogenesis and chondrogenesis of bone marrow stromal stem cells in alginate system

In orthopedics, the regeneration and repair of cartilage or bone defects after trauma, cancer, or metabolic disorders is still a major clinical challenge. Through developmental plasticity, bone marrow mesenchymal stem cells (BMSSCs) are important seed cells for the musculoskeletal tissue engineering approach. The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects.     

The Wnt antagonist Wif-1 interacts with CTGF and inhibits CTGF activity

Wnt inhibitory factor 1 (Wif-1) is a secreted antagonist of Wnt signalling. We recently demonstrated that this molecule is expressed predominantly in superficial layers of epiphyseal cartilage but also in bone and tendon. Moreover, we showed that Wif-1 is capable of binding to several cartilage-related Wnt ligands and interferes with Wnt3a-dependent Wnt signalling in chondrogenic cells. Here we provide evidence that the biological function of Wif-1 may not be confined to the modulation of Wnt signalling but appears to include the regulation of other signalling pathways. Thus, we show that Wif-1 physically binds to connective tissue growth factor (CTGF/CCN2) in vitro, predominantly by interaction with the C-terminal cysteine knot domain of CTGF. In vivo such an interaction appears also likely since the expression patterns of these two secreted proteins overlap in peripheral zones of epiphyseal cartilage. In chondrocytes CTGF has been shown to induce the expression of cartilage matrix genes such as aggrecan (Acan) and collagen2a1 (Col2a1). In this study we demonstrate that Wif-1 is capable to interfere with CTGF-dependent induction of Acan and Col2a1 gene expression in primary murine chondrocytes. Conversely, CTGF does not interfere with Wif-1-dependent inhibition of Wnt signalling. These results indicate that Wif-1 may be a multifunctional modulator of signalling pathways in the cartilage compartment.     

Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

Matrilin-1 is the prototypical member of the matrilin protein family and is highly expressed in cartilage. However, gene targeting of matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen IIα1a, indicating that the functional loss of this domain occurs as a consequence of matrilin-1 deficiency. The results show that matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events.     

Gene and protein expressions of type I collagen are regulated tissue-specifically in rat hyaline cartilages in vivo

The present study was designed to investigate how rat hyaline cartilages at various sites in vivo express the gene and protein of type I collagen using in situ hybridization and immunohistochemistry. The gene of pro alpha 1(I) collagen was expressed by chondrocytes in articular cartilage, and the protein of type I collagen was identified in the cartilage matrix. In contrast, growth plate cartilage expressed the gene of pro alpha 1(I) collagen, but no protein of type I collagen. Neither gene nor protein of type I collagen was expressed in cartilages of trachea and nasal septum. The present study suggested that expression of type I collagen in hyaline cartilages may be regulated tissue-specifically at gene and/or protein levels.