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While fibroblast growth factor (FGF) 23 is known as a phosphaturic factor in inherited and/or acquired hypophosphatemic disorders, it also serves an endocrine role in normal phosphate homeostasis. FGF23 acts negatively on the NaPi2a cotransporter and 25-hydroxy D(3)-1 alpha-hydroxylase with a resultant decrease in renal phosphate (Pi) reabsorption, while osteoblasts appear to be a primary source of FGF23 whose expression is counter-upregulated by 1 alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Here we have shown the distribution of FGF23 in normal rat bone and tooth, and its expression profile in fetal rat calvaria (RC) cell cultures. FGF23 mRNA was detectable in multiple fetal and adult tissues but levels were much higher in adult calvaria, femur and incisor, compared to the other tissues tested. Immunoreactive FGF23 was predominantly localized to osteoblasts, cementoblasts, and odontoblasts, with sporadic labeling in some chondrocytes, osteocytes and cementocytes. Notably, osteoclasts were also found to be a possible source of FGF23. Fetal bone and tooth germ cells labeled much less intensely than young adult osteoblasts and odontoblasts. In the RC cell model, FGF23 was expressed during osteoblast development. During matrix mineralization induced by beta-glycerophosphate (beta GP), FGF23 expression was transiently upregulated and then decreased to levels lower than in their non-beta GP-treated counterparts. 1,25(OH)(2)D(3) markedly increased FGF23 expression concomitant with the inhibition of beta GP-induced mineralization. Our data suggest that FGF23 expression in bone is closely correlated with bone formation in vitro and vivo, and points towards an important role(s) for FGF23 in young adult but not fetal mineralized tissues as a systemic factor for Pi homeostasis.

Accumulation and fragmentation of hyaluronic (HA) accompanies the inflammatory changes in the periodontium and gingival crevicular fluid are involved in periodontitis, but the mechanism for this is unknown. Recently, three human hyaluronan-synthase (HAS1, 2, and 3) genes have been cloned and characterised as synthesising hyaluronans of different molecular weights. Both HAS1 and HAS2 synthesise high molecular-weight HA, whereas HAS3 produces lower molecular weight HA. In the present study the regulation of HAS genes by cytokines in cultured human periodontal ligament (PDL) cells was investigated using a novel real-time fluorescence polymerase chain reaction detection system. Human PDL cells derived from premolars were cultured with or without tumour necrosing factor (TNF)-alpha (1-100 ng/ml), interleukin (IL)-1beta (0.1-10 ng/ml) and interferon (IFN)-gamma (1-100 ng/ml). Expression of HAS mRNA was assessed in cultured cells treated with these cytokines for 0-24 h. The expression of HAS2 mRNA was enhanced about 4.5- and 2.2-fold at maximum after 3-h stimulation with 10 ng/ml TNF-alpha and 1 ng/ml IL-1beta, respectively, whereas IFN-gamma exerted little effect on HAS2 or HAS3 mRNA expression during the experiment. Expression of HAS3 mRNA was increased by about 14- and 10-fold after 3-h stimulation with 10 ng/ml TNF-alpha and 1 ng/ml IL-1beta, respectively. These results suggest that TNF-alpha and IL-1beta regulate HAS expression, and consequently may result in an accumulation of HA and an increase in HA of a lower molecular-weight.

cDNAs for hyaluronic acid synthases (HAS2 and HAS3) were cloned from a cDNA library of cultured rabbit synovial membrane cells. The cDNA encoding the open reading frame of rabbit HAS2 and HAS3 was 1659 nucleotides in length with a predicted molecular mass of about 63 kDa. The amino acid sequence showed that the rabbit HAS2 was 98.7 and 98.4%, and HAS3 was 98.2 and 97.5% identical with human and mouse forms of the proteins, respectively. The predicted sequences for hyaluronic acid (HA) binding motifs and the catalytic domains related to beta 1-4 and beta 1-3 linkages, essential for HA synthesis, were almost conserved in both rabbit HAS2 and HAS3, similarly to human and mouse HASs. RT-PCR analysis and in situ hybridization revealed that the mRNA of HAS2 was highly expressed in the synovial membrane and articular cartilage, whereas the expression of HAS3 mRNA was slightest in these tissues. Thus, it is demonstrated that rabbit HASs are highly conserved in sequence content as compared to the human and mouse homologues described previously, and that HAS2 is predominantly expressed in the synovial membrane and articular cartilage, but HAS3 is not.

To elucidate the mechanism of accumulation and fragmentation of hyaluronic acid (HA) under inflammatory conditions, we investigated the effect of proinflammatory cytokines on hyaluronic acid synthetase (HAS) mRNA expression using cultured rabbit synovial membrane cells. HASs mRNA levels were determined by real-time PCR. HAS2 mRNA expression was maximally enhanced 3.3- and 2.8-fold after 3-hour stimulation with IL-1beta (1 ng/ml) and after 1-hour stimulation with TNF-alpha (10 ng/ml). HAS3 mRNA expression was increased by a maximum of 4.3 times after 3-hour stimulation with IL-1beta (10 ng/ml), whereas 1-hour stimulation with TNF-alpha (10 ng/ml) and IFN-gamma (10 ng/ml) induced around a 2.5-fold increase in HAS3 mRNA. Although IFN-gamma (1-100 ng/ml) alone showed little effect on HAS2 mRNA expression, the effect was synergized by combined with both IL-1beta and TNF-alpha, substantially increasing HAS2 mRNA expression. These results suggest that proinflammatory cytokines regulate the HAS expression, and consequently may contribute to the accumulation and fragmentation of HA.

Excessive mechanical load is thought to be responsible for the onset of osteoarthrosis (OA), but the mechanisms of cartilage destruction caused by mechanical loads remain unknown. In this study we applied a high magnitude cyclic tensile load to cultured chondrocytes using a Flexercell strain unit, which produces a change in cell morphology from a polygonal to spindle-like shape, and examined the protein level of cartilage matrixes and the gene expression of matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs) and proinflammatory cytokines such as IL-1beta and TNF-alpha. Toluidine blue staining, type II collagen immunostaining, and an assay of the incorporation of [35S]sulfate into proteoglycans revealed a decrease in the level of cartilage-specific matrixes in chondrocyte cultures subjected to high magnitude cyclic tensile load. PCR-Southern blot analysis showed that the high magnitude cyclic tensile load increased the mRNA level of MMP-1, MMP-3, MMP-9, IL-1beta, TNF-alpha and TIMP-1 in the cultured chondrocytes, while the mRNA level of MMP-2 and TIMP-2 was unchanged. Moreover, the induction of MMP-1, MMP-3 and MMP-9 mRNA expression was observed in the presence of cycloheximide, an inhibitor of protein synthesis. These findings suggest that excessive mechanical load directly changes the metabolism of cartilage by reducing the matrix components and causing a quantitative imbalance between MMPs and TIMPs.

Hyaluronan is a major component of the extracellular matrix of periodontal ligament (PDL) contributing to the structural and functional integrity. Hyaluronans contribute to the buffering effect of the PDL during chewing, and they are also important in inflammation and wound healing. Hyaluronan is known to be synthesized and turned over by the resident PDL cells, although the mechanisms of hyaluronan metabolism still remain unclear. Hyaluronidase (HAase), an endoglycosidase, degrades hyaluronan into small fragments. Currently, 3 human HAases, HYAL1, HYAL2, and PH-20, have been identified and well characterized.This study was conducted to investigate the expression and activity of these HAases in cultured human PDL fibroblasts and to elucidate the mechanisms involved in hyaluronan metabolism under normal and inflammatory conditions. Human PDL fibroblasts derived from the periodontium of 3 premolars were cultured with or without interleukin (IL)-1beta (0.1 to 10 ng/ml) and tumor necrosis factor (TNF)-alpha (1 to 100 ng/ml) for 0 to 48 hours. The expression of HAase mRNA was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR, and the enzymatic activity was examined using hyaluronan zymography.PDL fibroblasts expressed HYAL1 and HYAL2 mRNAs, but not PH-20 mRNA. The expression of HYAL1 mRNA was enhanced by about 3.5- and 3.7-fold at maximum after 1-hour stimulation with 1 ng/ml IL-1beta and after 3-hour stimulation with 10 ng/ml TNF-alpha, respectively. The expression of HYAL2 and PH-20 mRNAs was not affected by stimulation with cytokines. HAase activity was detected in conditioned medium from PDL fibroblast cultures, and the activity was enhanced by treatment with 10 ng/ml TNF-alpha.These results suggest that PDL fibroblasts express HAases and generate HAase activity essential for extracellular hyaluronan metabolism under physiological and inflammatory conditions.

In our previous studies, RGD-CAP/βig-h3 was isolated from a fiber-rich fraction of cartilage and was found to have a negative function on mineralization of hypertrophic chondrocytes. However, the expression and biological function of RGD-CAP in the periodontal ligament (PDL) are not known. We hypothesized that RGD-CAP could be expressed in the PDL and regulate its mineralization. To test this, we investigated the expression of RGD-CAP in human PDL and the effects of RGD-CAP on mineralization of cultured PDL cells. RGD-CAP was detected in the human PDL as multimeric proteins greater than 200 kDa. The RGD-CAP mRNA level decreased in cultured PDL cells exposed to 10−8 M dexamethasone or 10−8 M 1α,25-dihydroxyvitamin D3 when these steroids increased alkaline phosphatase (ALP) activity. Furthermore, exogenous RGD-CAP suppressed the ALP activity and bone nodule formation of cultured PDL cells. These findings suggest that RGD-CAP in the PDL modulates the mineralization which affects adjacent alveolar bone metabolism.