Dexamethasone downregulates the expression of parathyroid hormone-related protein (PTHrP) in mesenchymal stem cells.

Parathyroid hormone-related protein (PTHrP) has been shown to have anabolic effects in women with postmenopausal osteoporosis. PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The receptor responsible for the effects of PTHrP is the common PTH/PTHrP receptor (PTH1R). Glucocorticoids (GC) are commonly used as drugs to treat inflammatory diseases. Long-term GC treatments are often associated with bone loss which can lead to GC-induced osteoporosis. The aim of this work was to study the effects of the glucocorticoid dexamethasone (Dex) on the expression of PTHrP and PTH1R in adult human mesenchymal stem cells, the progenitor cells of osteoblasts. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The expression of PTHrP and PTH1R mRNA was assayed by real-time qPCR. The PTHrP release into the culture media was measured by an immunoradiometric assay. Treatment with Dex (10 nM) resulted in an 80% drop in the PTHrP release within 6 h. A 24 h Dex treatment also reduced the expression of PTHrP mRNA by up to 90%. The expression of PTH1R receptor mRNA was simultaneously increased up to 20-fold by 10 nM Dex. The effects of Dex on PTHrP and PTH1R were dose-dependent and experiments with the GC-receptor antagonist mifepristone showed an involvement of GC-receptors in these effects. In addition to the Dex-induced effects on PTHrP and PTH1R, Dex also increased mineralization and the expression of the osteoblast markers Runx2 and alkaline phosphatase. In our studies, we show that dexamethasone decreases the expression of PTHrP and increases the expression of the PTH1R receptor. This could have an impact on PTHrP-mediated anabolic actions on bone and could also affect the responsiveness of circulating PTH. The results indicate that glucocorticoids affect the signalling pathway of PTHrP by regulating both PTHrP and PTH1R expression and these mechanisms could be involved in glucocorticoid-induced osteoporosis.

Effects of phosphodiesterase 7 inhibition by RNA interference on the gene expression and differentiation of human mesenchymal stem cell-derived osteoblasts.

The second messenger molecule cyclic adenosine monophosphate (cAMP) plays an important role in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families designated PDE 1-11. The aim of this study was to investigate the effect of PDE7 and PDE8 inhibition on the gene expression and differentiation of human osteoblasts. Osteoblasts differentiated from human mesenchymal stem cells (hMSC) were cultured and treated with short interfering RNAs (siRNAs) generated from PDE7 and PDE8 PCR products. Total RNA was isolated from the cells, and gene expression was assayed with cDNA microarray and quantitative real-time PCR. bALP measurements were assayed during differentiation, and mineralization was determined by quantitative Alizarin red S staining. PDE7 and PDE8 inhibition by RNA interference decreased the gene expression of PDE7A by 60-70%, PDE7B by 40-50%, and PDE8A by 30%. PDE7 silencing increased the expression of beta-catenin, osteocalcin, caspase-8, and cAMP-responsive element-binding protein 5 (CREB-5) genes and decreased the expression of the 1, 25-dihydroxyvitamin D3 receptor gene. PDE8A silencing increased the expression of anti-apoptotic genes, but decreased the expression of osteoglycin (osteoinductive factor) and bone morphogenetic protein 1 (BMP-1). PDE7 silencing increased bALP and mineralization up to three-fold compared to controls. Treatment with the PDE7-selective PDE inhibitor BRL-50481 had similar effects on mineralization as the gene silencing. The PDE7 silencing also increased forskolin stimulated cAMP response, but had no effect on the proliferation rate. Furthermore, osteocalcin expression was increased by PDE7 silencing by a mechanism dependent on protein kinase A. Our results show that specific gene silencing with the RNAi method is a useful tool for inhibiting the gene expression of specific PDEs and that PDE7 silencing upregulates several osteogenic genes and increases mineralization. PDE7 may play an important role in the regulation of osteoblastic differentiation.

Long-term effects of tripeptide Ile-Pro-Pro on osteoblast differentiation in vitro.

Bone mineralization is a result of the function of bone-forming osteoblasts. Osteoblast differentiation from their precursors is a carefully controlled process that is affected by many signaling molecules. Protein-rich food-derived bioactive peptides are reported to express a variety of functions in vivo. We studied the long-term in vitro effect of bioactive tripeptide Ile-Pro-Pro (IPP) on osteoblasts differentiated from human mesenchymal stem cells. Osteoblast bone alkaline phosphatase activity (bALP), bone-forming capacity and gene expression were investigated. Treatment with 50 microM IPP had no effect on bALP activity, but osteoblast mineralization was increased. Gene expression of beta-catenin, Cbfa1/Runx2, PTHrP, CREB-5, osteoglycin, osteocalcin, caspase-8, osteoprotegerin (OPG) and RANKL was analyzed by quantitative real-time PCR on Days 13, 17 and 20 of culture. The results indicate that IPP increased mineral formation due to enhanced cell survival and matrix formation. In addition, IPP reduced the RANKL/OPG ratio. Bioactive peptides, such as IPP, could be one method by which a protein-rich diet promotes bone integrity.

Extracellular calcium regulates parathyroid hormone-related peptide expression in osteoblasts and osteoblast progenitor cells.

Parathyroid hormone-related peptide (PTHrP) has been shown to have anabolic effects on bone in women with postmenopausal osteoporosis. On the cellular level PTHrP promotes the recruitment of osteogenic cells and prevents apoptotic death of osteoblasts and osteocytes. The calcium concentration is considerably higher in the vicinity of resorbing osteoclasts than in the plasma. Therefore the osteoblasts are likely to be confronted by elevated extracellular calcium concentrations in the areas of resorptive activity. The present study was designed to assess the possibility that extracellular calcium could regulate PTHrP expression in osteoblastic cells. Adult human mesenchymal stem cells (hMSC) were cultured and differentiated by standard methods. The PTHrP release into the culture media was measured by an immunoradiometric assay and the expression of PTHrP, osteocalcin and Runx2 mRNA was assayed by real-time PCR. Increasing the extracellular calcium from 1 mM to 5 mM for 24 h resulted in a 4-6-fold increase in the PTHrP release. PTHrP mRNA was also increased by elevated calcium levels. The effect of calcium stimulation on PTHrP release could be seen within 60 min of treatment. The extracellular calcium sensing receptor (CaR) agonist neomycin mimicked the effects of calcium and the MEK/MAPK inhibitor PD98059 abolished the effect of calcium and neomycin. High extracellular calcium increased the mineralization of hMSC and the expression of osteocalcin, but this effect was not mimicked by neomycin. Our results show that in hMSC, elevated extracellular calcium levels increases both released PTHrP and PTHrP mRNA expression. The effect of calcium on PTHrP can be mimicked by activation of the CaR and can be diminished by inhibition of the MAPK signalling pathway.

Effects of bioactive peptides isoleucine-proline-proline (IPP), valine-proline-proline (VPP) and leucine-lysine-proline (LKP) on gene expression of osteoblasts differentiated from human mesenchymal stem cells.

Food-derived bioactive peptides are reported to express a variety of functions in vivo. We studied the in vitro effect of three bioactive tripeptides, isoleucine-proline-proline (IPP), valine-proline-proline (VPP) and leucine-lysine-proline (LKP), on osteoblast proliferation and gene expression. We used UMR-106 osteosarcoma cells, human marrow-derived mesenchymal stem cells (hMSC) and osteoblasts differentiated from hMSC. Treatment with 50 mum-IPP increased UMR-106 cell and hMSC proliferation. The gene expression of hMSC-differentiated osteoblasts was analysed by the microarray method. Microarray analysis revealed that IPP up-regulated 270 genes and down-regulated 100 genes. VPP and LKP, by contrast, had a very modest influence on osteoblast gene expression. Real-time PCR confirmed that IPP up-regulated PTHrP, BMP-5 and CREB-5 and down-regulated VDR and caspase-8. IPP possesses potential to increase osteoblast proliferation, differentiation and signalling. Agents that increase the number and function of osteoblasts could improve bone mass and structure, and decrease fracture risk.

Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation.

The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A.

Dexamethasone down-regulates cAMP-phosphodiesterase in human osteosarcoma cells.

Cyclic adenosine monophosphate (cAMP) is an important second messenger in the hormonal regulation of bone metabolism. cAMP is inactivated by the cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 known families, designated PDE1-11. Interference with the cAMP signaling pathway has been suggested as one mechanism causing glucocorticoid induced osteoporosis. We speculated that glucocorticoids could affect the cAMP pathway by a down-regulation of PDE-mediated cAMP hydrolysis. The main cAMP hydrolysing enzyme families of human MG-63 and SaOS-2 osteosarcoma cells were identified as PDE1 and PDE4 by assaying the PDE activity of Q-sepharose fractions and cell homogenates with selective inhibitors. Treatment with the glucocorticoid dexamethasone (Dex) decreased cAMP-PDE activity by up to 50%, without affecting cGMP-PDE activity. Dex treatment reduced the sensitivity of the total cAMP-PDE activity towards the PDE4 selective PDE inhibitor rolipram. Forskolin stimulated cAMP accumulation was increased 30-60-fold in the presence of rolipram. Treatment with Dex did not affect the basal or forskolin stimulated cAMP accumulation, but treatment resulted in a reduced effect of rolipram on cAMP accumulation. Expression of the following cAMP-PDE subtypes were detected by reverse transcriptase PCR (RT-PCR): PDE1A, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, PDE7A, PDE7B, PDE8A, PDE10A and PDE11A. Using semi-quantitative RT-PCR, we detected a 50-70% decrease in the mRNA of PDE4A and PDE4B subtypes following Dex treatment. Further analysis revealed that Dex reduced the PDE4A4 and PDE4B1 isoforms. PDE4A1 PDE4A, PDE4A7, PDE4A10, PDE4B2 were also expressed, but Dex did not affect the transcription of these isoforms. We conclude that Dex treatment could affect the cAMP signaling pathway of human osteosarcoma cells by reducing type 4 cAMP-phosphodiesterase (PDE4).