Mikael Ahlström

Rapid Protein Kinase A—Mediated Activation of Cyclic AMP‐Phosphodiesterase by Parathyroid Hormone in UMR‐106 Osteoblast‐like Cells

Parathyroid hormone (PTH) plays an essential role in osteoblast proliferation and differentiation. The effects of PTH are known to be mediated by cyclic adenosine monophosphate (cAMP) and calcium and by the activation of protein kinase C (PKC). cAMP is hydrolyzed to the inactive form 5′ AMP by cyclic nucleotide phosphodiesterases (PDEs). We have investigated the role of PTH on PDE regulation in UMR‐106 osteoblast‐like cells. Treatment with 10 nM PTH caused a 3‐fold increase in the PDE activity. The activation of PDE could be seen within 2 minutes and reached maximal levels after 20 minutes. The PTH effect was dose dependent with a half‐maximal dose of 2 nM. The effect of PTH could be mimicked by the cAMP analogs Bt2 cAMP and forskolin, but not by PTH fragment 3–34, calcium ionophore A23187, or by the PKC activator phorbol 12‐myristate 13‐acetate. The PDE activity stimulated by PTH could be abolished by the PKA inhibitor H‐8. The PDE activated by PTH was inhibitable by low concentrations of the cAMP‐PDE–specific inhibitor RO 20‐1724 (IC50 = 0.2 μM), but not by low concentrations of the inhibitors of cGMP‐stimulated and cGMP‐inhibited PDEs MEP‐1 and milrinone (IC50 for both compounds >30 μM). The PTH‐stimulated cAMP accumulation was potentiated about 7‐fold in the presence of RO 20‐1724. H‐8 potentiated the PTH‐stimulated cAMP accumulation about 4‐fold. Our results show that PTH rapidly stimulates the activity of cAMP‐PDE in UMR‐106 cells. The PDE activation involves cAMP and PKA. Inhibition of PKA can abolish the PTH‐stimulated PDE activation and leads to increased accumulation of intracellular cAMP.

Sphingosine 1-phosphate mobilizes sequestered calcium, activates calcium entry, and stimulates deoxyribonucleic acid synthesis in thyroid FRTL-5 cells.

Sphingosine 1-phosphate (SPP) potently mobilizes sequestered calcium and is a mitogen in several cell types. In the present investigation, we have evaluated the effect of SPP on intracellular free calcium concentration ([Ca2+]i) and synthesis of DNA in thyroid FRTL-5 cells. SPP rapidly and transiently mobilized sequestered calcium and stimulated entry of extracellular calcium. The entry of calcium, but not the mobilization, was in part inhibited by pretreatment with pertussis toxin (Ptx), and by activation of protein kinase C. SPP did not stimulate the production of inositol 1,4,5-trisphosphate. SPP stimulated the incorporation of 3H-thymidine in a time- and dose-dependent manner. The effect was not inhibited by Ptx. Furthermore, SPP stimulated the activation of the proto-oncogene c-fos. SPP rapidly tyrosine-phosphorylated an approximately 66 kDa protein. This phosphorylation persisted for at least 1 h. Pretreatment of the cells with genistein abolished the SPP-evoked tyrosine phosphorylation, and attenuated the SPP-evoked increase in [Ca2+]i. Furthermore, the SPP-evoked activation of Na+-H+ exchange was inhibited by genistein. The phosphorylation was not attenuated by pretreatment of the cells with Ptx. SPP per se did not affect cellular cAMP levels but attenuated the TSH-evoked increase in cAMP. As the effect of SPP might be due to activation of phospholipase D, we tested whether phosphatidic acid (PA) mobilized calcium or stimulated the incorporation of 3H-thymidine. PA mobilized sequestered calcium but did not stimulate calcium entry. PA very modestly enhanced the incorporation of 3H-thymidine. Our results suggest, that SPP stimulates DNA synthesis and activates entry of calcium in FRTL-5 cells. The effect on calcium entry appears to be dependent, at least in part, on one or several tyrosine kinases.

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.

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.

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.

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.

1,25-Dihydroxyvitamin D3 reduces the number of α1-andrenergic receptors in FRTL-5 rat thyroid cells

Abstract Noradrenaline and ATP evokes a transient increase in the intracellular calcium concentration ([Ca2+]i) in FRTL-5 cells. In a previous study, we showed that 1,25-dihydroxyvitamin-D3 (1,25(OH)2-D3) increases the ATP evoked changes in [Ca2+]i. In the present paper, we found that pre-incubating the cells with 10 nM 1,25(OH)2-D3 for 48 h did not affect the noradrenaline-evoked increase in [Ca2+]i. We subsequently examined if this could be due to an effect of 1,25(OH)2-D3 on α1-adrenergic receptor number, or receptor affinity. Pretreatment with 10 nM 1,25(OH)2-D3 for 48 h decreased the binding of the α1-adrenergic specific antagonist [3H]prazosin by 55% (Bmax for 1,25(OH)2-D3 treated = 27.6 ± 5.0 fmol/mg protein, untreated = 61.7 ± 5.4 fmol/mg protein). No effect of 1,25(OH)2-D3 on the affinity for [3H]prazosin was observed. The effect of 1,25(OH)2-D3 on the [3H]prazosin binding was both time- and dose-dependent and could first be seen after 8–12 h of 1,25(OH)2-D3 treatment, indicating a genomic effect. The effect of 1,25(OH)2-D3 could be abolished with the protein synthesis inhibitor cycloheximide. No effect on the [3H]prazosin binding could be seen after a 48 h preincubation with 100 nM of either 24,25-dihydroxyvitamin D3 and 25-dihydroxyvitamin D3, indicating that the effect of 1,25(OH)2-D3 was specific. The cellular cAMP concentration was decreased after 48 h treatment with 10 nM 1,25(OH)2-D3. When TSH was replaced with dibutyryl cAMP or forskolin the [3H]prazosin binding increased. 1,25(OH)2-D3 also reduced the dibutyryl cAMP and forskolin stimulated [3H]prazosin binding. In addition, 1,25(OH)2-D3 reduced the [3H]prazosin binding in TSH deficient media. Our results suggest that 1,25(OH)2-D3 reduces the number of α1-adrenergic receptors via a dual mechanism: by reducing cAMP production and by affecting some mechanism(s) downstream from the production of cAMP.