Treatment Of Bone Cells Research Articles

The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas Receptor axis as a key player in alveolar bone remodeling.

The renin-angiotensin system (RAS), aside its classical hormonal properties, has been implicated in the pathogenesis of inflammatory disorders. The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas Receptor (ACE2/Ang-(1-7)/MasR) axis owns anti-inflammatory properties and was recently associated with bone remodeling in osteoporosis. Thus, the aim of this study was to characterize the presence and effects of the ACE2/Ang-(1-7)/MasR axis in osteoblasts and osteoclasts in vitro and in vivo. ACE2 and MasR were detected by qPCR and western blotting in primary osteoblast and osteoclast cell cultures. Cells were incubated with different concentrations of Ang-(1-7), diminazene aceturate (DIZE - an ACE2 activator), A-779 (MasR antagonist) and/or LPS in order to evaluate osteoblast alkaline phosphatase and mineralized matrix, osteoclast differentiation and cytokine expression, and mRNA levels of osteoblasts and osteoclasts markers. An experimental model of alveolar bone resorption triggered by dysbiosis in rats was used to evaluate bone remodeling in vivo. Rats were treated with Ang-(1-7), DIZE and/or A-779 and periodontal samples were collected for immunohistochemistry, morphometric analysis, osteoblast and osteoclast count and cytokine evaluation. Human gingival samples from healthy and periodontitis patients were also evaluated for detection of ACE2 and MasR expression. Osteoblasts and osteoclasts expressed ACE2 and MasR in vitro and in vivo. LPS stimulation or alveolar bone loss induction reduced ACE2 expression. Treatment of bone cells with Ang-(1-7) or DIZE stimulated osteoblast ALP, matrix synthesis, upregulated osterix, osteocalcin and collagen type 1 transcription, reduced IL-6 expression, and decreased osteoclast differentiation, RANK and IL-1β mRNA transcripts, and IL-6 and IL-1β levels, in a MasR-dependent manner. In vivo, Ang-(1-7) and DIZE decreased alveolar bone loss through improvement of osteoblast/osteoclast ratio. A-779 reversed such phenotype. ACE2/Ang-(1-7)/MasR axis activation reduced IL-6 expression, but not IL-1β. ACE2 and MasR were also detected in human gingival samples, with higher expression in the healthy than in the inflamed tissues. These findings show that the ACE2/Ang-(1-7)/MasR is an active player in alveolar bone remodeling.

Shedding of a soluble form of BMP receptor-IB controls bone cell responses to BMP

Bone morphogenetic proteins (BMP) are members of the transforming growth factor β (TGF-β) superfamily and are involved in a wide variety of biological processes, including osteoblast differentiation and bone healing. The activities of the BMP are mediated by signal transduction via three BMP receptors (BMPR-IA, -IB and -II), which are thus essential for the biological actions of the BMP. Although the precise mechanisms which control the BMPR are not yet known, it is possible that post-translational regulation of these cell surface antigens by shedding could modulate their expression and thereby at least partly determine the response of the cells to the BMP. To test this possibility, the present study has examined whether soluble forms of the BMPR are produced by shedding from primary human bone cells in vitro. The results showed that human bone cells expressed both mRNA transcripts and antigens corresponding to BMPR-IA, -IB and -II. Incubation of the cells with phorbol 12-myristate 13-acetate (PMA), a potent inducer of proteolytic shedding, resulted in a pronounced decrease in cell surface expression of all three BMPR and, concurrently, the presence of ‘soluble’ forms of these antigens in culture supernatants. Moreover, PMA treatment significantly reduced the level of BMP-2-induced Smad1/5 phosphorylation, a major early activation step in signal transduction initiated by BMP/BMPR interaction. It is notable that, while treatment of bone cells with interleukin-1β (IL-1β) also reduced the level of surface BMPR-IB, this inflammatory cytokine had no effect on BMPR-IA or -II levels, hence only the soluble form of BMPR-IB was detected. Furthermore, in addition to down-regulating BMP-2-induced Smad1/5 phosphorylation, IL-1β also caused a reduction in the level of BMP-2-induced alkaline phosphatase activity and osteocalcin expression, both closely associated with bone cell differentiation. In conclusion, our study has provided evidence, for the first time, that BMPR can be modulated at the cell surface by the shedding of a soluble form of the antigen, resulting in a markedly diminished response to BMP-2 in vitro.

Initiation of Osteoclast Bone Resorption by Interstitial Collagenase

Osteoclasts form an acidic compartment at their attachment site in which bone demineralization and matrix degradation occur. Although both the cysteine proteinases and neutral collagenases participate in bone resorption, their roles have remained unclear. Here we show that interstitial collagenase has an essential role in initiating bone resorption, distinct from that of the cysteine proteinases. Treatment of osteoclasts with cysteine proteinase inhibitors did not affect the number of resorption lacunae ("pits") formed on the surface of dentine slices, but it generated abnormal pits that were demineralized but filled with undegraded matrix. Treatment with metalloproteinase inhibitors did not alter the qualitative features of lacunae, but it greatly reduced the number of pits and surface area resorbed. Treatment of bone cells with an inhibitory anti-rat interstitial collagenase antiserum reduced bone resorption markedly. In the presence of collagenase inhibitors, resorption was restored by pretreatment of dentine slices with rat interstitial collagenase or by precoating the dentine slices with collagenase-derived gelatin peptides or heat-gelatinized collagen. Immunostaining revealed that interstitial collagenase is produced at high levels by stromal cells and osteoblasts adjacent to osteoclasts. These results indicate that interstitial collagenase can function as a "coupling factor," allowing osteoblasts to initiate bone resorption by generating collagen fragments that activate osteoclasts.

Stimulation of uridine incorporation in isolated bone cells by parathyroid hormone and cyclic AMP.

Treatment of isolated bone cells with purified bovine parathyroid hormone (5 ng/ml-2 ¼g/ml) augmented the incorporation of 214C-uridine into acid-soluble RNA precursor pools and into RNA. Stimulation developed within 15 min and gradually declined after 30 min. A synthetic N-terminal 1–34 amino acid fragment of PTH was one-half as potent as native hormone. A 2–34 amino acid fragment, oxidized native hormone, and several basic polypeptides were inactive. PTH enhanced the labeling of cellular UDP and UTP pools appreciably and increased that of UMP slightly but failed to modify the total radioactivity of cellular free uridine, pointing to uridine or UMP phosphorylation (or both) as the site(s) of PTH action. Stimulation of 2–14C-uridine incorporation was not associated withalterations in total cell ATP levels. The effect of PTH on uridine metabolism appeared during simultaneous treatment of bone cells with actinomycin D, indicating that it was independent of RNAsynthesis. Exogenous (Bt)2 cAMP (0.1–1.0 min) an...

Impaired Accumulation of Ribonucleic Acid Precursors and Depletion of Ribonucleic Acid in Glucocorticoid-treated Bone Cells

Abstract We have examined the effects of cortisol in vitro on RNA metabolism in isolated, cultured bone cells. Treatment for as little as 2 hours decreased the incorporation of radioactively labeled nucleosides (uridine, adenosine, or guanosine) into RNA. This appears to result from reduced influx or phosphorylation (or both) of free nucleosides, since the accumulation of radioactivity in intracellular nucleotide pools (UMP, UDP, UTP) was also inhibited, but efflux of uridine nucleotides was not increased. Inhibition persisted after refeeding treated cells with fresh, cortisol-free medium, and was evident at a high (1 mm) as well as a low (13 µm) starting concentration of uridine in the incubation medium, suggesting that it was not caused by enhanced dilution of radioactive uridine in expanded intra- or extracellular pools of nonradioactive uridine. The possibility that the accumulation of radioactivity in nucleotide pools was inhibited by a feedback mechanism caused by prior reduction in RNA synthesis seems unlikely, in view of the simultaneous and equivalent effects of cortisol on the labeling of nucleotide pools and RNA. Treatment of bone cells with actinomycin D was accompanied by a decrease in the accumulation of RNA precursors only after inhibition of RNA synthesis was well established. Protein synthesis may be required for this response to cortisol since cortisol was ineffective when cells were treated simultaneously with either puromycin or cycloheximide, inhibitors of protein synthesis which did not in themselves block nucleoside incorporation. In addition to inhibiting the accumulation of RNA precursors and their incorporation into RNA, cortisol treatment gradually reduced the total content of RNA per culture (5 to 10% in 5 hours, 20 to 30% in 15 hours) and the amount of 18 S and 28 S ribosomal RNA per culture. Two observations suggest that this loss of RNA results in part from an acceleration of RNA breakdown: (a) the breakdown of endogenous, previously labeled RNA was increased in isolated microsomes derived from cortisol-treated cells and (b) addition of cortisol to cells which had been continuously labeled with radioactive uridine from initial seeding until treatment significantly enhanced the decline in RNA total radioactivity during the chase period. In addition, the rate of RNA synthesis, as estimated from the change in RNA specific radioactivity during the chase period, was decreased by treatment with cortisol. Cortisol effects were related to its glucocorticoid activity, and were directly proportional to its starting concentration, minimal changes appearing at near physiological concentrations (10 to 100 mµm).