Hyaluronidase expression in cultured growth plate chondrocytes during differentiation

Hyaluronan (HA) is a major component of the extracellular matrix of cartilage, contributes to its structural and functional integrity, and has various important roles in the differentiation of chondrocytes. HA metabolism is regulated by both anabolic and catabolic processes; however, the details have not yet been clarified. The purpose of this study was to clarify the expression patterns of hyaluronidase (HAase) mRNAs (from the relevant HAase genes: the HYALs) and HAase activity during chondrocyte differentiation. Cartilage tissue and growth plate chondrocytes were isolated from the ribs of 4-week-old male Japanese rabbits. The expression of HYAL mRNAs in cartilage was analyzed by in situ hybridization. The expression levels of HYAL mRNAs in the culture were analyzed for each of the chondrocyte differentiation stages by means of quantitative real-time polymerase chain reaction analysis. Enzymatic activity in the conditioned medium from the cultures was examined by using HA zymography and an enzyme-linked immunosorbent-like assay. The expression levels of HYAL1 and HYAL2 mRNAs were enhanced about 2.8-fold and 3.2-fold at the maximum during the early matrix forming stage, respectively, and by about 3.2-fold and 2.0-fold at the maximum in the hypertrophic stage, respectively. HYAL3 mRNA was not detected throughout the experimental period. HAase activity was enhanced at the early matrix forming and hypertrophic stages. These results suggest that selective expression of HYALs is essential for extracellular HA metabolism during chondrocyte differentiation.This research was supported by Grants-in-Aid (no. 11557166) for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.     

Chondrocyte protein with a poly-proline region is a novel protein expressed by chondrocytes in vitro and in vivo

Chondrogenic differentiation is a multistep process entailing the sequential activation and inhibition of the expression of a number of genes. To identify genes preferentially expressed at the hypertrophic stage rather than early differentiation stages of chicken chondrocyte differentiation, a subtracted cDNA library was generated. Here we describe the characterization of a cDNA isolated from this library and that of the encoded protein referred to as Chondrocyte Protein with a Poly-proline Region (CHPPR).The cDNA coding for CHPPR hybridizes with a 3.0-kb mRNA expressed at extremely low levels in dedifferentiated chondrocytes, cultured in adherent conditions, at low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes in suspension culture. The Parathyroid Hormone peptide [PTH (1-34)] enhances accumulation of CHPPR mRNA in cultured chondrocytes. This 3.0-kb mRNA is also detectable in several chick embryo tissues but at a lower extent when compared to that present in cartilage and in hypertrophic chondrocytes. The CHPPR cDNA has a complete open reading frame coding for a polypeptide with a calculated mass of 35.6 kDa containing a proline-rich region with a PPLP motif (single-letter amino acid code). We demonstrate by Western blot analysis that two CHPPR isoforms are detected in the cell lysates from cultured chondrocytes when they are not in the culture medium; furthermore, we find that the CHPPR gene is expressed in vivo by chick embryo chondrocytes at higher levels in the prehypertrophic and hypertrophic zones.     

Phosphate-induced Apoptosis of Hypertrophic Chondrocytes Is Associated with a Decrease in Mitochondrial Membrane Potential and Is Dependent upon Erk1/2 Phosphorylation

Growth plate abnormalities, associated with impaired hypertrophic chondrocyte apoptosis, are observed in humans and animals with abnormalities of vitamin D action and renal phosphate reabsorption. Low circulating phosphate levels impair hypertrophic chondrocyte apoptosis, whereas treatment of these cells with phosphate activates the mitochondrial apoptotic pathway. Because phosphate-mediated apoptosis of chondrocytes is differentiation-dependent, studies were performed to identify factors that contribute to hypertrophic chondrocyte apoptosis. An increase in the percentage of cells with low mitochondrial membrane potential, evaluated by JC-1 fluorescence, was observed during hypertrophic differentiation of primary murine chondrocytes in culture. This percentage was further increased by treatment of hypertrophic, but not proliferative, chondrocytes with phosphate. Phosphate-mediated apoptosis was observed as early as 30 min post-treatment and was dependent upon Erk1/2 phosphorylation. Inhibition of Erk1/2 phosphorylation in vivo confirmed an important role for this signaling pathway in regulating hypertrophic chondrocyte apoptosis in growing mice. Murine embryonic metatarsals cultured under phosphate-restricted conditions demonstrated a 2.5-fold increase in parathyroid hormone-related protein mRNA expression accompanied by a marked attenuation in phospho-Erk immunoreactivity in hypertrophic chondrocytes. Thus, these investigations point to an important role for phosphate in regulating mitochondrial membrane potential in hypertrophic chondrocytes and growth plate maturation by the parathyroid hormone-related protein signaling pathway.     

Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals

Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on chondrocytes, we characterized primary chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled "Bio-Stretch" device. Cyclic matrix deformation greatly stimulated proliferation of immature chondrocytes, but not that of hypertrophic chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.     

Phosphate regulates embryonic endochondral bone development

Phosphate is required for terminal differentiation of hypertrophic chondrocytes during postnatal growth plate maturation. In vitro models of chondrocyte differentiation demonstrate that 7 mM phosphate, a concentration analogous to that of the late gestational fetus, activates the mitochondrial apoptotic pathway in hypertrophic chondrocytes. This raises the question as to whether extracellular phosphate modulates chondrocyte differentiation and apoptosis during embryonic endochondral bone formation. To address this question, we performed investigations in the mouse metatarsal culture model that recapitulates in vivo bone development. Metatarsals were cultured for 4, 8, and 12 days with 1.25 and 7 mM phosphate. Metatarsals cultured with 7 mM phosphate showed a decrease in proliferation compared to those cultured in 1.25 mM phosphate. This decrease in proliferation was accompanied by an early enhancement in hypertrophic chondrocyte differentiation, associated with an increase in FGF18 expression. By 8 days in culture, an increase caspase‐9 activation and apoptosis of hypertrophic chondrocytes was observed in the metatarsals cultured in 7 mM phosphate. Immunohistochemical analyses of embryonic bones demonstrated activation of caspase‐9 in hypertrophic chondrocytes, associated with vascular invasion. Thus, these investigations demonstrate that phosphate promotes chondrocyte differentiation during embryonic development and implicate a physiological role for phosphate activation of the mitochondrial apoptotic pathway during embryonic endochondral bone formation. J. Cell. Biochem. 108: 668–674, 2009. © 2009 Wiley‐Liss, Inc.     

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

The growth arrest and DNA damage-inducible 45beta (GADD45beta) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45beta in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45beta as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45beta mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45beta protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45beta(-/-) mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45beta in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45beta stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45beta plays an essential role during chondrocyte terminal differentiation.     

Concentration of mRNA for the natriuretic peptide receptor-C in hypertrophic chondrocytes of the fetal mouse tibia

The roles of natriuretic peptides in cardiovascular homeostasis have been well characterized. A recent study revealed that mice lacking natriuretic peptide receptor-C (NPR-C) exhibit skeletal-overgrowth. We therefore, performed in situ hybridization with riboprobes to determine the localization of mRNAs for receptors for natriuretic peptides in the growth plate of the fetal mouse tibia The amount of mRNA for NPR-A was below the detectable level in the growth plate. The mRNA for NPR-B was detected predominantly in proliferating chondrocytes. By contrast, high levels of mRNA for NPR-C were found in hypertrophic chondrocytes. In other regions of the growth plate, the levels of mRNA for NPR-C were very low. The patterns of expression of mRNAs for NPR-B and NPR-C, namely, subtype switching during differentiation from proliferating chondrocytes to hypertrophic chondrocytes, suggest that these receptors might be involved in the growth and differentiation of the growth plate during fetal development in the mouse.     

Transforming growth factor-beta 1, -beta 2 and -beta 3 in cartilage and bone cells during endochondral ossification in the chick.

The localization of TGF-beta 1, -beta 2 and -beta 3 was studied in the growth plate, epiphysis and metaphysis of the tibiotarsus of three-week-old chicks. The different TGF-beta isoforms were localized to hypertrophic chondrocytes, chondroclasts, osteoblasts and osteoclasts using immunohistochemical staining analysis with specific TGF-beta antibodies. TGF-betas in osteoclasts and chondroclasts were restricted to those cells located on the respective matrices. TGF-beta 3 localization was mainly cytoplasmic in the transitional (early hypertrophic) chondrocytes, but nuclear staining was also detected in some proliferating chondrocytes. The cell-specific localization of these TGF-beta isoforms supports the hypothesis that TGF-beta has a role in the coupling of new bone formation to bone and cartilage matrix resorption during osteochondral development and suggests that TGF-beta may be a marker of chondrocyte differentiation. TGF-beta localization preceded a marked increase in type II collagen mRNA expression in transitional chondrocytes, suggesting a role for TGF-beta in the induction of synthesis of extracellular matrix.     

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.     

Expression of hyaluronan synthase 1 and distribution of hyaluronan during follicular atresia in pig ovaries

CD44 on macrophages is recognized as a phagocytic receptor involved in the phagocytosis of apoptotic cells. Recently, we detected CD44 on macrophages in atretic follicles during atresia. In this study, we evaluated the distribution of the principal CD44 ligand hyaluronan (HA) and the expressions of HA synthases (HAS: HAS1, HAS2, and HAS3) during atresia in pig ovaries. We determined the 2139-bp sequence of Sus scrofa HAS1 and raised an anti-HAS1 polyclonal antibody. The S. scrofa HAS1 sequence contained six putative HA-binding motifs and conserved amino acid residues crucial for GlcNac transferase activity. HAS1 mRNA expression was upregulated during atresia; however, HAS2 and HAS3 mRNA expression levels were low and very low to undetectable, respectively. Western blotting showed that HAS1 was markedly upregulated during atresia. Immunohistochemical analyses revealed HAS1 distribution in theca cells of healthy and early atretic (stages I and II) follicles and in progressing atretic (stage III) follicles. Hyaluronan was visualized with the HA-binding protein; it accumulated in the theca layer during all stages and in stage III follicles. Hyaluronan assay showed a significantly increased HA concentration in follicular fluid at stage III. Flow cytometry showed HAS1 expression in 55.7% of SIRPA-positive macrophages in stage III follicles. Our results suggest that the HA concentration in follicular fluids increased during atresia and that HAS1 may be the dominant HAS protein in theca cells to produce HA in pig ovaries.