Extracellular localization of galectin-3 has a deleterious role in joint tissues

In this study we examine the extracellular role of galectin-3 (gal-3) in joint tissues. Following intra-articular injection of gal-3 or vehicle in knee joints of mice, histological evaluation of articular cartilage and subchondral bone was performed. Further studies were then performed using human osteoarthritic (OA) chondrocytes and subchondral bone osteoblasts, in which the effect of gal-3 (0 to 10 μg/ml) was analyzed. Osteoblasts were incubated in the presence of vitamin D₃ (50 nM), which is an inducer of osteocalcin, encoded by an osteoblast terminal differentiation gene. Genes of interest mainly expressed in either chondrocytes or osteoblasts were analyzed with real-time RT-PCR and enzyme immunoassays. Signalling pathways regulating osteocalcin were analyzed in the presence of gal-3. Intra-articular injection of gal-3 induced knee swelling and lesions in both cartilage and subchondral bone. On human OA chondrocytes, gal-3 at 1 μg/ml stimulated ADAMTS-5 expression in chondrocytes and, at higher concentrations (5 and 10 μg/ml), matrix metalloproteinase-3 expression. Experiments performed with osteoblasts showed a weak but bipolar effect on alkaline phosphatase expression: stimulation at 1 μg/ml or inhibition at 10 μg/ml. In the absence of vitamin D₃, type I collagen alpha 1 chain expression was inhibited by 10 μg/ml of gal-3. The vitamin D₃induced osteocalcin was strongly inhibited in a dose-dependent manner in the presence of gal-3, at both the mRNA and protein levels. This inhibition was mainly mediated by phosphatidylinositol-3-kinase. These findings indicate that high levels of extracellular gal-3, which could be encountered locally during the inflammatory process, have deleterious effects in both cartilage and subchondral bone tissues.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E₂ (PGE₂) or leukotriene B₄ (LTB₄) by subchondral osteoblasts, PGE₂ levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE₂ to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB₄ levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)₂D₃ and TGF-β also modulated PGE₂ production. TGF-β stimulated PGE₂ production in both OA osteoblast groups, whereas 1,25(OH)₂D₃ alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE₂ production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE₂ levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE₂ to LTB₄ is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)₂D₃ and TGF-β depending on their endogenous low and high PGE₂ levels.     

Effect of IL‐1β, PGE2, and TGF‐β1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)‐1β, proftaglandin E₂ (PGE₂), and transforming growth factor‐β1 (TGF‐β1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real‐time PCR. In all cultures, OPG mRNA increased significantly in response to IL‐1β treatment and decreased in response to TGF‐β1 whereas PGE₂ treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL‐1β treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF‐β1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption. J. Cell. Biochem. 110: 304–310, 2010. © 2010 Wiley‐Liss, Inc.     

RANKL synthesized by articular chondrocytes contributes to juxta-articular bone loss in chronic arthritis

Introduction

The receptor activator nuclear factor-kappaB ligand (RANKL) diffuses from articular cartilage to subchondral bone. However, the role of chondrocyte-synthesized RANKL in rheumatoid arthritis-associated juxta-articular bone loss has not yet been explored. This study aimed to determine whether RANKL produced by chondrocytes induces osteoclastogenesis and juxta-articular bone loss associated with chronic arthritis.

Methods

Chronic antigen-induced arthritis (AIA) was induced in New Zealand (NZ) rabbits. Osteoarthritis (OA) and control groups were simultaneously studied. Dual X-ray absorptiometry of subchondral knee bone was performed before sacrifice. Histological analysis and protein expression of RANKL and osteoprotegerin (OPG) were evaluated in joint tissues. Co-cultures of human OA articular chondrocytes with peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated with macrophage-colony stimulating factor (M-CSF) and prostaglandin E₂ (PGE₂), then further stained with tartrate-resistant acid phosphatase.

Results

Subchondral bone loss was confirmed in AIA rabbits when compared with controls. The expression of RANKL, OPG and RANKL/OPG ratio in cartilage were increased in AIA compared to control animals, although this pattern was not seen in synovium. Furthermore, RANKL expression and RANKL/OPG ratio were inversely related to subchondral bone mineral density. RANKL expression was observed throughout all cartilage zones of rabbits and was specially increased in the calcified cartilage of AIA animals. Co-cultures demonstrated that PGE₂-stimulated human chondrocytes, which produce RANKL, also induce osteoclasts differentiation from PBMCs.

Conclusions

Chondrocyte-synthesized RANKL may contribute to the development of juxta-articular osteoporosis associated with chronic arthritis, by enhancing osteoclastogenesis. These results point out a new mechanism of bone loss in patients with rheumatoid arthritis.     

The influence of weight loss on serum osteoprotegerin concentration in obese perimenopausal women

Objective: To assess the influence of weight reduction therapy on serum osteoprotegerin (OPG) concentration in obese patients and compare these results with normal‐weight controls. Research Methods and Procedures: Forty‐three obese women (BMI, 36.7 ± 4.1 kg/m²; mean age, 50.1 ± 4.5 years) were studied. The control group consisted of 19 normal‐weight women (BMI, 24.2 ± 2.1 kg/m²; mean age, 53.8 ± 5.2 years). In all patients, serum concentrations of OPG, C telopeptide of type I collagen containing the cross‐linking site (CTX), osteocalcin, parathormone, 25‐(OH)‐D₃ (vitamin D), and total calcium and phosphorus were assessed before and after a 3‐month weight reduction therapy. Results: In obese subjects, serum concentrations of OPG, 25‐(OH)‐D₃, osteocalcin, total calcium, and phosphorus were significantly lower, and serum concentration of parathormone was significantly higher, before weight reduction therapy in comparison with normal‐weight controls. After weight reduction, a significantly higher serum concentration of 25‐(OH)‐D₃ and CTX and significantly lower concentration of OPG were found. Discussion: Serum concentration of OPG was significantly lower in obese patients in comparison with normal‐weight controls. Weight reduction therapy resulted in further decrease in OPG serum concentrations. Therefore, OPG cannot be treated as a protective factor from bone loss in obese patients.     

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)₂ vitamin D₃ significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.     

Bone morphogenetic protein 2 enhances mouse osteoclast differentiation via increased levels of receptor activator of NF-κB ligand expression in osteoblasts

1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] induces osteoclast formation via induction of receptor activator of NF-κB ligand (RANKL, also called TNF-related activation-induced cytokine: TRANCE) in osteoblasts. In cocultures of mouse bone marrow cells and osteoblasts, 1,25(OH)₂D₃ induced osteoclast formation in a dose-dependent manner, with maximum osteoclast formation observed at concentrations greater than 10^?9 M of 1,25(OH)₂D₃. In the presence of bone morphogenetic protein 2 (BMP-2), the maximum formation of osteoclasts was seen with lower concentrations of 1,25(OH)₂D₃ (greater than 10^?11 M), suggesting that BMP-2 enhances osteoclast formation induced by 1,25(OH)₂D₃. In addition, the expressions of RANKL mRNA and proteins were induced by 1,25(OH)₂D₃ in osteoblasts, and further upregulated by BMP-2. In mouse bone marrow cell cultures without 1,25(OH)₂D₃, BMP-2 did not enhance osteoclast differentiation induced by recombinant RANKL and macrophage colony-stimulating factor (M-CSF), indicating that BMP-2 does not target osteoclast precursors. Furthermore, BMP-2 up-regulated the expression level of vitamin D receptor (VDR) in osteoblasts. These results suggest that BMP-2 regulates mouse osteoclast differentiation via upregulation of RANKL in osteoblasts induced by 1,25(OH)₂D₃.     

Targeting bone alleviates osteoarthritis in osteopenic mice and modulates cartilage catabolism

Objective

Subchondral bone modifications occur early in the development of osteoarthritis (OA). The level of bone resorption might impact cartilage remodeling. We therefore assessed the in vivo and in vitro effects of targeting bone resorption in OA and cartilage metabolism.

Methods

OA was induced by meniscectomy (MNX) in ovariectomized osteopenic mice (OP) treated with estradiol (E2), pamidronate (PAM), or phosphate buffered saline (PBS) for 6 weeks. We assessed the subchondral bone and cartilage structure and the expression of cartilage matrix proteases. To assess the involvement of bone soluble factors in cartilage metabolism, supernatant of human bone explants pre-treated with E2 or PAM were transferred to cartilage explants to assess proteoglycan release and aggrecan cleavage. OPG/RANKL mRNA expression was assessed in bone explants by real-time quantitative PCR. The role of osteoprotegerin (OPG) in the bone-cartilage crosstalk was tested using an OPG neutralizing antibody.

Results

Bone mineral density of OP mice and osteoclast number were restored by E2 and PAM (p<0.05). In OP mice, E2 and PAM decreased ADAMTS-4 and -5 expression, while only PAM markedly reduced OA compared to PBS (2.0±0.63 vs 5.2±0.95; p<0.05). OPG/RANKL mRNA was increased in human bone explants treated with both drugs (2.2–3.7-fold). Moreover, supernatants from bone explants cultured with E2 or PAM reduced aggrecan cleavage and cartilage proteoglycan release (73±8.0% and 80±22% of control, respectively, p<0.05). This effect was reversed with osteoprotegerin blockade.

Conclusion

The inhibition of bone resorption by pamidronate in osteopenic mice alleviates the histological OA score with a reduction in the expression of aggrecanases. Bone soluble factors, such as osteoprotegerin, impact the cartilage response to catabolic factors. This study further highlights the importance of subchondral bone in the regulation of joint cartilage damage in OA.     

Baicalein alleviates osteoarthritis by protecting subchondral bone,inhibiting angiogenesis and synovial proliferation

Osteoarthritis (OA) is one of the most frequent chronic joint diseases with the increasing life expectancy. The main characteristics of the disease are loss of articular cartilage, subchondral bone sclerosis and synovium inflammation. Physical measures, drug therapy and surgery are the mainstay of treatments for OA, whereas drug therapies are mainly limited to analgesics, glucocorticoids, hyaluronic acids and some alternative therapies because of single therapeutic target of OA joints. Baicalein, a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi, has been widely used in anti-inflammatory therapies. Previous studies revealed that baicalein could alleviate cartilage degeneration effectively by acting on articular chondrocytes. However, the mechanisms involved in baicalein-mediated protection of the OA are not completely understood in consideration of integrality of arthrosis. In this study, we found that intra-articular injection of baicalein ameliorated subchondral bone remodelling. Further studies showed that baicalein could decrease the number of differentiated osteoblasts by inhibiting pre-osteoblasts proliferation and promoting pre-osteoblasts apoptosis. In addition, baicalein impaired angiogenesis of endothelial cells and inhibited proliferation of synovial cells. Taken together, these results implicated that baicalein might be an effective medicine for treating OA by regulating multiple targets.     

Kruppel-like factor 4 attenuates osteoblast formation,function, and cross talk with osteoclasts

Osteoblasts not only control bone formation but also support osteoclast differentiation. Here we show the involvement of Kruppel-like factor 4 (KLF4) in the differentiation of osteoclasts and osteoblasts. KLF4 was down-regulated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in osteoblasts. Overexpression of KLF4 in osteoblasts attenuated 1,25(OH)₂D₃-induced osteoclast differentiation in co-culture of mouse bone marrow cells and osteoblasts through the down-regulation of receptor activator of nuclear factor κB ligand (RANKL) expression. Direct binding of KLF4 to the RANKL promoter repressed 1,25(OH)₂D₃-induced RANKL expression by preventing vitamin D receptor from binding to the RANKL promoter region. In contrast, ectopic overexpression of KLF4 in osteoblasts attenuated osteoblast differentiation and mineralization. KLF4 interacted directly with Runx2 and inhibited the expression of its target genes. Moreover, mice with conditional knockout of KLF4 in osteoblasts showed markedly increased bone mass caused by enhanced bone formation despite increased osteoclast activity. Thus, our data suggest that KLF4 controls bone homeostasis by negatively regulating both osteoclast and osteoblast differentiation.     

CCL20/CCR6 chemokine/receptor expression in bone tissue from osteoarthritis and rheumatoid arthritis patients: Different response of osteoblasts in the two groups

Subchondral bone remodeling in osteoarthritis (OA) and rheumatoid arthritis (RA) is mainly characterized by the formation of osteophytes/fibrosis and by the presence of infiltrating cells associated to bone resorption. In this study we analyzed CC (cysteine cysteine motif) chemokine ligand (CCL)20 and CC chemokine receptor (CCR)6 function in subchondral bone tissue and osteoblasts isolated from OA and RA patients. CCL20/CCR6 expression was evaluated by immunohistochemical techniques in bone tissue from OA and RA patients. CCL20‐functional tests were performed on osteoblasts isolated from OA and RA patients to evaluate enzymatic response and cell proliferation. Moreover, we assessed Akt phosphorylation as the major signaling pathway for CCL20. In bone tissue biopsies we found that osteoblasts from both OA and RA patients expressed CCR6 while CCL20 was expressed only by RA osteoblasts. Both CCR6 and CCL20 were highly expressed in osteocytes and mononuclear cells from only RA patients. CCL20‐stimulated OA osteoblasts showed a significant increase in β‐N‐acetylhexosaminidase release compared to RA. Conversely, a significant increase in cellular proliferation was found only in CCL20‐stimulated RA osteoblasts associated to Akt phosphorylation. These data were confirmed in bone tissue biopsies. This study demonstrates a different expression of CCL20‐positive osteoblasts in OA versus RA disease that seem to be associated with the presence of infiltrating mononuclear cells. Moreover, CCL20 stimulation resulted in a greater proliferative response in RA osteoblasts compared to OA osteoblasts, mediated by Akt signaling, while OA osteoblasts showed increased enzymatic activity, thus suggesting a differential role of this chemokine in OA and RA. J. Cell. Physiol. 221: 154–160, 2009. © 2009 Wiley‐Liss, Inc     

Effects of high glucose and high insulin concentrations on osteoblast function in vitro

Bone disease as a consequence of diabetes mellitus (DM) is not fully understood. The effects of high glucose (30 mM), high insulin (50 nM), or mannitol (30 mM; osmotic control) were evaluated on MC3T3-E1 cells (osteoblasts) in vitro. The mRNA and protein levels of parathyroid hormone (PTH) receptor (PTH1R), collagen I, RANKL, osteoprotegerin (OPG), alkaline phosphatase (ALP), and glucose transporter (GLUT1) were estimated by real-time polymerase chain reaction or Western blotting. The mineralization capacity was analyzed by von Kossa staining. High glucose induced overexpression of RANKL (2×) and OPG (30×), suggesting that RANKL-induced osteoclast activity might not be a dominant mechanism of bone disease in DM, since this increase was followed by increased OPG. Collagen I increased by 12×, indicating an excess of organic matrix production. The expression of ALP decreased by 50 %, indicating a deficit in mineralization capacity, confirmed by von Kossa staining. Mannitol induced similar effects as glucose suggesting that extracellular hyperosmolarity was able to stimulate organic matrix production. GLUT1 expression was not altered, and insulin did not reverse most of the effects of glucose, suggesting that glucose uptake by osteoblasts was not altered by high glucose. The data suggest that the bone fragility typical of DM is not a consequence of excessive bone reabsorption but is instead attributable to a defect in organic matrix mineralization. The heightened increase in OPG versus RANKL might cause a decrease in the bone-remodeling cycle. Osteoblasts appear to be more sensitive to extracellular hypertonicity than to the intracellular metabolic effects of hyperglycemia.     

Genistein aglycone effect on bone loss is not enhanced by supplemental calcium and vitamin D3: A dose ranging experimental study

Genistein aglycone (GEN) has a favorable effect on bone loss. We investigated the effects of GEN alone or in combination with supplemental calcium and vitamin D₃ in an animal model of bone loss to evaluate if there was additional benefit. Ovariectomized (OVX) and SHAM-OVX rats were used. OVX were divided into 12 groups and randomized to receive: GEN at 27, 54, 200, 500 or 1000 mg (human equivalent dose (HED)/day/ip injection alone or with calcium carbonate (Ca) (360 mg/kg/day/gavages) and vitamin D₃ (D₃) (50 IU/kg/day/gavages) or Ca/D₃ without GEN or untreated for 6 weeks. SHAM-OVX were randomized into 7 groups and treated with: Ca and D₃ alone or in combination with GEN (same doses as OVX), or left untreated. Bone mineral density (BMD), bone-alkaline phosphatase (b-ALP), collagen C-telopeptides (CTX), osteoprotegerin (OPG) and soluble receptor activator of NFκB ligand (sRANKL) were assessed. Femurs were excised and tested for breaking strength and histology. Uterine weight was analyzed to assess GEN's estrogenic effects on the SHAM-OVX.The most effective dose of GEN, independent of Ca/D₃ supplementation, was 54 mg/day. Higher doses yielded no further improvement in bone biomarkers, histology or strength. Only 1000 mg/day HED of genistein produced statistically significant changes in uterine weight of the SHAM-OVX. This study suggests that 54 mg/day of GEN is the threshold dose for efficacy. In addition, supplemental calcium and vitamin D₃, beyond normal dietary intake do not enhance the effects of genistein on improving measures of bone loss. This observation has implications regarding the use of calcium and vitamin D₃ supplementation.     

Application of <Emphasis Type="Italic">in vivo</Emphasis> micro-computed tomography in the temporal characterisation of subchondral bone architecture in a rat model of low-dose monosodium iodoacetate-induced osteoarthritis

Introduction  

Osteoarthritis (OA) is a complex, multifactorial joint disease affecting both the cartilage and the subchondral bone. Animal models of OA aid in the understanding of the pathogenesis of OA and testing suitable drugs for OA treatment. In this study we characterized the temporal changes in the tibial subchondral bone architecture in a rat model of low-dose monosodium iodoacetate (MIA)-induced OA using in vivo micro-computed tomography (CT).     

1,25,26-trihydroxyvitamin D3: isolation, identification, and biological activity

A polar metabolite of vitamin D₃ has been produced in vitro from either 1,25-dihydroxyvitamin D₃ incubated with kidney homogenate from vitamin D-supplemented chickens or from 25,26-dihydroxyvitamin D₃ incubated with vitamin D-deficient chicken kidney homogenate. This compound was isolated in pure form and identified as 1,25,26-trihydroxyvitamin D₃ by ultraviolet absorption spectrophotometry and mass spectrometry. Furthermore, its periodate cleavage product comigrates with synthetic 1α-hydroxy-25-keto-27-norvitamin D₃ on high-performance liquid chromatography. The 1,25,26-trihydroxyvitamin D₃ is 0.1-0.01 as active as 1,25-dihydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone calcium mobilization.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

1,25-dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status

Our laboratory has recently demonstrated that 1,25-dihydroxyvitamin D₃(1,25(OH)₂D₃) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D-sufficient rat colonocytes. These effects of 1,25(OH)₂D₃ were, however, lost in vitamin D-insufficient rats and restored by the in vivo repletion of 1,25(OH)₂D₃. In the present studies we have examined the ability of 1,25(OH)₂D₃ to stimulate the phosphorylation of colonic membrane proteins in intact D-sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)₂D₃ increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D-sufficient rats. Moreover, in vitamin D-sufficient rats, treatment of colonocytes with 1,25(OH)₂D₃ or 12-Otertradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)₂D₃ were both rapid and transient. In addition, PKC_19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)₂D₃. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D-sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D-insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)₂D₃-treatment of colonocytes from vitamin D-insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)₂D₃ following the in vivo repletion of vitamin D-insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)₂D₃ treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley-Liss, Inc.     

Treatment with ephrin B2 positively impacts the abnormal metabolism of human osteoarthritic chondrocytes

Introduction  

Members of the ephrin system, the ephrin receptor erythropoietin-producing hepatocellular B4 (EphB4) and its specific ligand, ephrin B2, appear to be involved in the bone remodelling process. We recently showed that their interaction inhibits the resorptive activity of human osteoarthritic (OA) subchondral bone osteoblasts. Hence, we further investigated the possible implication of these ephrin members on the catabolic/anabolic activities of human OA chondrocytes.