Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Osteopontin-deficient bone cells are defective in their ability to produce NO in response to pulsatile fluid flow

We have previously identified a mutation (R273W) in the von Willebrand factor (VWF) propeptide that results in quantitative deficiency of plasma VWF and a loss of high molecular weight VWF multimers. Recombinant VWF having the R273W mutation (rVWFR273W) expressed in COS-7 cells demonstrated severely impaired secretion and degradation in an intracellular location [Allen, S., et al. (2000) Blood 96, 560-568]. In this report we used pulse-chase analysis and endoglycosidase H digestion of wild-type rVWF and rVWFR273W immunoprecipitated from COS-7 cells to show that rVWFR273W was retained in the endoplasmic reticulum (ER). We demonstrate for the first time that wild-type rVWF and rVWFR273W interacted with the thiol-dependent oxidoreductase ERp57 during biosynthesis in the ER. Pulse chase analysis demonstrated that the interactions of rVWFR273W with ERp57 and calnexin were prolonged compared to wild-type rVWF. In contrast there was no apparent difference between rVWFR273W and wild-type rVWF in their time-courses of interaction with calreticulin.     

Estrogen enhances mechanical stress-induced prostaglandin production by bone cells from elderly women

Several studies indicate that estrogen may enhance the effects of mechanical loading on bone mineral density in elderly women. This stimulating effect of estrogen could be due to increased sensitivity of bone cells to mechanical stress in the presence of estrogen. The present study was performed to determine whether 17beta-estradiol (E2) enhances mechanical stress-induced prostaglandin production and cyclooxygenase (COX)-2 mRNA expression. We subjected bone cells from seven nonosteoporotic women between 56 and 75 yr of age for 1 h to pulsating fluid flow (PFF) in the presence or absence of 10(-11) M E2 and measured prostaglandin production and COX-1 and COX-2 mRNA expression. One hour of PFF stimulated prostaglandin (PGE2) production threefold, PGI2 production twofold, and COX-2, but not COX-1, mRNA expression 2.9-fold. Addition of E2 further enhanced PFF-stimulated PGE2 production by 1.9-fold but did not significantly affect PGI2 production or COX-2 or COX-1 mRNA expression. E2 by itself did not affect any of the parameters measured. These results suggest that estrogen modulates bone cell mechanosensitivity via the prostaglandin synthetic pathway independently of COX mRNA expression.     

Nitric oxide production by bone cells is fluid shear stress rate dependent

Shear stress due to mechanical loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. Moreover, the rate (determined by frequency and magnitude) of mechanical loading determines the amount of bone formation. Whether the bone cells' response to fluid shear stress is rate dependent is unknown. Here we investigated whether bone cell activation by fluid shear stress is rate dependent. MC3T3-E1 osteoblastic cells were subjected for 15 min to fluid shear stress of varying frequencies and amplitudes, resulting in peak fluid shear stress rates ranging from 0 to 39.6 Pa-Hz. Nitric oxide production, a parameter for bone cell activation, was found to be linearly dependent on the fluid shear stress rate; the slope was steepest at 5 min (0.11 Pa-Hz(-1)) and decreased to 0.03 Pa-Hz(-1) at 15 min. We conclude that the fluid shear stress rate is an important parameter for bone cell activation.     

The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts

Fluid flowing through the bone porosity might be a primary stimulus for functional adaptation of bone. Osteoblasts, and osteocytes in particular, respond to fluid flow in vitro with enhanced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release; both of these signaling molecules mediate mechanically-induced bone formation. Because the cell cytoskeleton is involved in signal transduction, we hypothesized that the pulsatile fluid flow-induced release of NO and PGE(2) in both osteoblastic and osteocytic cells involves the actin and microtubule cytoskeleton. In testing this hypothesis we found that fluid flow-induced NO response in osteoblasts was accompanied by parallel alignment of stress fibers, whereas PGE(2) response was related to fluid flow stimulation of focal adhesions formed after cytoskeletal disruption. Fluid flow-induced PGE(2) response in osteocytes was inhibited by cytoskeletal disruption, whereas in osteoblasts it was enhanced. These opposite PGE(2) responses are likely related to differences in cytoskeletal composition (osteocyte structure was more dependent on actin), but may occur via cytoskeletal modulation of shear/stretch-sensitive ion channels that are known to be dominant in osteocyte (and not osteoblast) response to mechanical loading.     

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.     

Differential stimulation of prostaglandin G/H synthase-2 in osteocytes and other osteogenic cells by pulsating fluid flow

Mechanical stress produces flow of fluid in the osteocytic lacunar-canalicular network, which is likely the physiological signal for the adaptive response of bone. We compared the induction of prostaglandin G/H synthase-2 (PGHS-2) by pulsating fluid flow (PFF) and serum in osteocytes, osteoblasts, and periosteal fibroblasts, isolated from 18-day-old fetal chicken calvariae. A serum-deprived mixed population of primarily osteocytes and osteoblasts responded to serum with a two- to threefold induction of PGHS-2 mRNA. Serum stimulated PGHS-2-derived PGE(2) release from osteoblasts and osteocytes but not from periosteal fibroblasts as NS-398, a PGHS-2 blocker, inhibited PGE(2) release from osteocytes and osteoblasts with 65%, but not that from periosteal fibroblasts. On the other hand PFF (0.7 Pa, 5 Hz) stimulated (3 fold) PGHS-2 mRNA only in OCY. The related PGE(2) response could be completely inhibited by NS-398. We conclude that osteocytes have a higher intrinsic sensitivity for loading-derived fluid flow than osteoblasts or periosteal fibroblasts.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.     

A new autosomal recessive form of Stickler syndrome is caused by a mutation in the COL9A1 gene

Stickler syndrome is characterized by ophthalmic, articular, orofacial, and auditory manifestations. It has an autosomal dominant inheritance pattern and is caused by mutations in COL2A1, COL11A1, and COL11A2. We describe a family of Moroccan origin that consists of four children with Stickler syndrome, six unaffected children, and two unaffected parents who are distant relatives (fifth degree). All family members were clinically investigated for ear, nose, and throat; ophthalmologic; and radiological abnormalities. Four children showed symptoms characteristic of Stickler syndrome, including moderate-to-severe sensorineural hearing loss, moderate-to-high myopia with vitreoretinopathy, and epiphyseal dysplasia. We considered the COL9A1 gene, located on chromosome 6q13, to be a candidate gene on the basis of the structural association with collagen types II and XI and because of the high expression in the human inner ear indicated by cDNA microarray. Mutation analysis of the coding region of the COL9A1 gene showed a homozygous R295X mutation in the four affected children. The parents and four unaffected children were heterozygous carriers of the R295X mutation. Two unaffected children were homozygous for the wild-type allele. None of the family members except the homozygous R295X carriers had any signs of Stickler syndrome. Therefore, COL9A1 is the fourth identified gene that can cause Stickler syndrome. In contrast to the three previously reported Stickler syndrome-causing genes, this gene causes a form of Stickler syndrome with an autosomal recessive inheritance pattern. This finding will have a major impact on the genetic counseling of patients with Stickler syndrome and on the understanding of the pathophysiology of collagens. Mutation analysis of this gene is recommended in patients with Stickler syndrome with possible autosomal recessive inheritance.