Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

Independent and interrelated regulation of ornithine decarboxylase by calcium and cAMP in fetal rat osteoblasts

The present study was undertaken to determine in fetal rat osteoblasts whether and how the intracellular messengers calcium and cAMP are involved in stimulation of ornithine decarboxylase (ODC) activity. For that purpose we used different drugs affecting [Ca2+]i and cAMP concentration. A23187 stimulates ODC activity in a biphasic way, with maximal stimulation at 100 nM A23187. At that concentration no stimulation of cAMP production was observed. Basal and A23187-stimulated (100 nM) ODC activity were inhibited by EGTA and trifluoperazine. Forskolin stimulated dose-dependently both ODC activity and cAMP production. Besides these effects forskolin (1 and 10 microM) increased the [Ca2+]i via an increased calcium influx. Addition of La3+, verapamil or EGTA, but not of trifluoperazine, significantly inhibited the forskolin-stimulated (10 microM) ODC activity. When forskolin (100 nM and 1 microM) was added together with 1 microM A23187, a synergistic stimulation of ODC activity was observed. These results implicate that calcium is involved in basal ODC activity, and that ODC activity can be stimulated via (1) a cAMP-independent calcium pathway, and (2) a calcium-dependent, cAMP pathway. It is proposed that ODC activity can be stimulated via interaction between calcium and cAMP.     

Phorbol ester induces desensitization of PTH-stimulated cyclic AMP production by decreasing the PTH receptor binding in UMR-106 cells

Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with the protein kinase C(PK-C) activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) results in a time dependent (1-12h) desensitization of PTH-stimulated cAMP production. Compared to controls, PMA-treated cells showed 50% decrease of PTH-stimulated cAMP production. PK-C inhibitor, H-7 significantly blocked this PMA-induced desensitization. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was decreased by about 20% in PMA-treated cells. H-7 treatment completely restored receptor binding in PMA-treated cells. These data suggest that PK-C might act directly on PTH receptor which is coupling to adenylate cyclase, and induce desensitization.     

Role of calcium and cAMP in heterologous up-regulation of the 1,25-dihydroxyvitamin D3 receptor in an osteoblast cell line.

To understand further the mechanism of action of parathyroid hormone (PTH) in the stimulation of the number of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) binding sites in UMR 106-01 cells we studied the role of cAMP and calcium. In addition to PTH other agents known to act via the cAMP signal pathway, prostaglandin E2, forskolin and dibutyryl cAMP, caused an increase in 1,25(OH)2D3 binding. Addition of the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl)adenine resulted in a marked decrease of PTH-stimulated cAMP production but this was not followed by a reduction of 1,25(OH)2D3 receptor up-regulation by PTH. Increasing the intracellular calcium concentration by Bay K 8644 and A23817 independent of an activation of the cAMP signal pathway did not result in an increased 1,25(OH)2D3 binding. The calcium channel blockers nitrendipine and verapamil and chelating extracellular calcium with EGTA all reduced cAMP-mediated stimulation of 1,25(OH)2D3 binding. This reduction was not due to a reduce cAMP production as verapamil even potentiated PTH- and forskolin-stimulated cAMP production in a dose-dependent manner. The present study provides evidence for an interrelated action of calcium and cAMP in the heterologous up-regulation of the 1,25(OH)2D3 receptor. The current data show an interaction between the cAMP and calcium signal pathway at (1) the level of cAMP generation/degradation, and (2) a level located distal in the cascade leading to 1,25(OH)2D3 receptor up-regulation.     

The role of protein kinase C-delta in PTH stimulation of IGF-binding protein-5 mRNA in UMR-106-01 cells

We have investigated the role of protein kinase C (PKC) signal transduction pathways in parathyroid hormone (PTH) regulation of insulin-like growth factor-binding protein-5 (IGFBP-5) gene expression in the rat osteoblast-like cell line UMR-106-01. Involvement of the PKC pathway was determined by the findings that bisindolylmaleimide I inhibited 40% of the PTH effect, and 1 microM bovine PTH-(3-34) stimulated a 10-fold induction of IGFBP-5 mRNA. PTH-(1-34) and PTH-(3-34) (100 nM) both stimulated PKC-delta translocation from the membrane to the nuclear fraction. Rottlerin, a PKC-delta-specific inhibitor, and a dominant negative mutant of PKC-delta were both able to significantly inhibit PTH-(1-34) and PTH-(3-34) induction of IGFBP-5 mRNA, suggesting a stimulatory role for PKC-delta in the effects of PTH. Phorbol 12-myristate 13-acetate (PMA) stimulated PKC-alpha translocation from the cytosol to the membrane and inhibited approximately 50% of the PTH-(1-34), forskolin, and 8-bromoadenosine 3',5'-cyclic monophosphate-stimulated IGFBP-5 mRNA levels, suggesting that PKC-alpha negatively regulates protein kinase A (PKA)-mediated induction of IGFBP-5 mRNA. These results suggest that the induction of IGFBP-5 by PTH is both PKA and PKC dependent and PKC-delta is the primary mediator of the effects of PTH via the PKC pathway.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Parathyroid hormone potentiates nucleotide-induced [Ca2+]i release in rat osteoblasts independently of Gq activation or cyclic monophosphate accumulation. A mechanism for localizing systemic responses in bone

The regulation of tissue turnover requires the coordinated activity of both local and systemic factors. Nucleotides exist transiently in the extracellular environment, where they serve as ligands to P2 receptors. Here we report that the localized release of these nucleotides can sensitize osteoblasts to the activity of systemic factors. We have investigated the ability of parathyroid hormone (PTH), a principal regulator of bone resorption and formation, to potentiate signals arising from nucleotide stimulation of UMR-106 clonal rat osteoblasts. PTH receptor activation alone did not lead to [Ca(2+)](i) elevation in these cells, indicating no G(q) coupling, however, activation of G(q)-coupled P2Y(1) receptors resulted in characteristic [Ca(2+)](i) release. PTH potentiated this nucleotide-induced Ca(2+) release, independently of Ca(2+) influx. PTH-(1-31), which activates only G(s), mimicked the actions of PTH-(1-34), whereas PTH-(3-34), which only activates G(q), was unable to potentiate nucleotide-induced [Ca(2+)](i) release. Despite this coupling of the PTHR to G(s), cAMP accumulation or protein kinase A activation did not contribute to the potentiation. 3-Isobutyl-1-methylxanthine, but not forskolin effectively potentiated nucleotide-induced [Ca(2+)](i) release, however, further experiments proved that cyclic monophosphates were not involved in the potentiation mechanism. Costimulation of UMR-106 cells with P2Y(1) agonists and PTH led to increased levels of cAMP response element-binding protein phosphorylation and a synergistic effect was observed on endogenous c-fos gene expression following costimulation. In fact the calcium responsive Ca/cAMP response element of the c-fos promoter alone was effective at driving this synergistic gene expression. These findings demonstrate that nucleotides can provide a targeted response to systemic factors, such as PTH, and have important implications for PTH-induced signaling in bone.     

Role of increase in intracellular calcium in PTH-induced homologous desensitization in UMR-106 cells.

Effects of increase in intracellular calcium on PTH-induced homologous desensitization were investigated using calcium ionophores. Pretreatment of UMR-106 cells (rat osteoblast like osteosarcoma cell line) with calcium ionophores (A23187 or ionomycin) for 6h resulted in approximately 50% decrease of PTH-stimulated cAMP production. PTH receptor binding, assessed with 125I-[Nle8,Nle18,Tyr34]PTH-(1-34) as radioligand, was significantly decreased in 10(-6) M calcium ionophore-pretreated (for 6h) cells without affecting the dissociation constant (Kd) for PTH. Minimal effective treatment period was 2h and similar inhibitory effect was observed in 12h-treated cells. These data suggest that increase in intracellular calcium might also act on PTH receptor in the similar manner as protein kinase C activation to induce desensitization.     

The influence of calcium ionophore and activators of protein kinase C on steroid production by preovulatory ovarian follicles of the goldfish

Steroidogenesis in teleost fish, as in other vertebrate groups, is mediated by the activation of adenylate cyclase. For the present studies, calcium ionophore A23187 and either phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were used to investigate the possible roles that changes in intracellular calcium content and protein kinase C activation play in steroid production by goldfish preovulatory ovarian follicles incubated in vitro. While ineffective alone, PMA (1.6-400 nM) and OAG (25-100 micrograms/ml) exhibited classical synergism with A23187 (1.0-10 microM), leading to increased testosterone production. The magnitude of these responses was at least tenfold lower than that obtained with human chorionic gonadotropin (hCG), forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Testosterone production stimulated by hCG and forskolin was blocked by addition of PMA but not OAG. Unlike PMA, the inactive phorbol ester 4 alpha-phorbol 12,13-dideconate did not influence basal or stimulated testosterone production. A23187 had a biphasic effect on stimulated testosterone production: a dosage of 0.25 or 1.0 microM potentiated the action of submaximally effective dosages of hCG or forskolin on testosterone production; a higher dosage of 4 microM inhibited stimulated testosterone production by up to 50%. In conclusion, these studies suggest that, in addition to the adenylate cyclase second messenger system, changes in intracellular calcium and activation of protein kinase C may modulate steroidogenesis in goldfish ovarian follicles.     

Thyrotropin stimulates progesterone secretion by luteal cells by activation of the cAMP/protein kinase A signaling system: a potential involvement of protein kinase C

Although the corpus luteum (CL) is not known as a target tissue for thyrotropin (TSH), this hormone increases progesterone production by porcine luteal cells cultured in vitro. In this study we investigated the optimal conditions for TSH-stimulated progesterone secretion as well as the involvement of protein kinase A (PKA) and protein kinase C (PKC) in the mechanism of TSH action on porcine luteal cells. To study the PKA and PKC signaling mechanisms, luteal cells collected from mature CL were incubated with the inhibitor of PKA and potent activators of both kinases: PKA-forskolin and PKC-phorbol ester 12-myriistate-13-acetate (PMA). The PKA inhibitor totally suppressed progesterone production in TSH alone, forskolin alone and in TSH plus forskolin-stimulated luteal cells. Forskolin increased basal (P < 0.05) and TSH-stimulated (P < 0.05) progesterone secretion and cAMP accumulation (P < 0.05). Forskolin and PMA added together to control (non-TSH-treated) luteal cells had an additive effect on progesterone production. In TSH-treated cells, the effect of PMA was statistically significant but did not show an additive effect with forskolin. Further PMA did not affect cAMP accumulation in control and TSH-treated luteal cells. Treatment of control and TSH-treated luteal cells with forskolin and PMA together showed the same increase in cAMP accumulation as with forskolin alone. This is the first demonstration that TSH acts on luteal cell steroidogenesis by activation of the cAMP/PKA second messenger system and also that the PKC signaling pathway may be involved in luteal TSH action on the corpus luteum.     

Stimulation of extracellular signal-regulated kinases and proliferation in rat osteoblastic cells by parathyroid hormone is protein kinase C-dependent

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation.     

Parathyroid hormone and parathyroid hormone-related protein exert both pro- and anti-apoptotic effects in mesenchymal cells

During bone formation, multipotential mesenchymal cells proliferate and differentiate into osteoblasts, and subsequently many die because of apoptosis. Evidence suggests that the receptor for parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP), the PTH-1 receptor (PTH-1R), plays an important role in this process. Multipotential mesenchymal cells (C3H10T1/2) transfected with normal or mutant PTH-1Rs and MC3T3-E1 osteoblastic cells were used to explore the roles of PTH, PTHrP, and the PTH-1R in cell viability relative to osteoblastic differentiation. Overexpression of wild-type PTH-1R increased cell numbers and promoted osteocalcin gene expression versus inactivated mutant receptors. Furthermore, the effects of PTH and PTHrP on apoptosis were dramatically dependent on cell status. In preconfluent C3H10T1/2 and MC3T3-E1 cells, PTH and PTHrP protected against dexamethasone-induced reduction in cell viability, which was dependent on cAMP activation. Conversely, PTH and PTHrP resulted in reduced cell viability in postconfluent cells, which was also dependent on cAMP activation. Further, the proapoptotic-like effects were associated with an inhibition of Akt phosphorylation. These data suggest that parathyroid hormones accelerate turnover of osteoblasts by promoting cell viability early and promoting cell departure from the differentiation program later in their developmental scheme. Both of these actions occur at least in part via the protein kinase A pathway.     

Regulation of the corpus luteum by protein kinase C. I. Phosphorylation activity and steroidogenic action in large and small ovine luteal cells

The activity and steroidogenic action of protein kinase C were evaluated in small and large steroidogenic ovine luteal cells. Protein kinase C activity (per mg protein) was threefold greater in large than in small luteal cells, whereas protein kinase A activity was similar in the two cell types. Phorbol 12-myristate 13-acetate (PMA) activated protein kinase C in luteal cells as demonstrated by membrane association of 91% of available protein kinase C within 15 min of PMA treatment. Longer treatments with PMA produced cells with low protein kinase C activity (protein kinase C-deficient cells) but did not affect cellular viability or protein kinase A activity. Activation of protein kinase C caused an acute, dose-dependent inhibition of progesterone production in unstimulated large and luteinizing hormone (LH)-stimulated small luteal cells. This inhibition by PMA appeared to be specific for protein kinase C since it was greatly attenuated in protein kinase C-deficient cells and since an inactive phorbol ester, 4 alpha-phorbol, had no effect on luteal progesterone production. The inhibitory locus of protein kinase C action in small luteal cells appeared to be distal to the adenylate cyclase enzyme because progesterone production was inhibited similarly in cells stimulated with LH, forskolin, or dibutyryl cyclic adenosine 3',5'-monophosphate. Cholesterol side-chain cleavage activity, as measured by metabolism of 25-hydroxycholesterol, was inhibited by PMA in large, but not in small, luteal cells. These data indicate that activation of protein kinase C specifically inhibits progesterone production in both large and small ovine luteal cells, although the intracellular mechanisms invoked appear to differ in the two cell types.     

Cross-talk of parathyroid hormone-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Its role in PTH-induced homologous desensitization of intracellular calcium response

The present study was designed to characterize the cross-talk of parathyroid hormone (PTH)-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [PKC]) and its participation in PTH-induced homologous desensitization of intracellular calcium ([Ca²⁺]i) in osteoblastic UMR-106 cells. Although our recent study revealed that prolonged (more than 2 h) pretreatment with PKC-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) significantly decreased the PTH-stimulated cAMP production, pretreatment with PMA (10^?7 and 10^?6 M) but not 10^?6 M 4alphaphorbol 12,13-didecanoate (PDD), incapable of activating PKC for 30 min significantly augmented 10^?7 M hPTH-(1-34)-stimulated cAMP production. H-7 (50 uM), a PKC inhibitor, significantly antagonized this PMA-induced effect. Pretreatment with 10^?6 M PMA for 30 min did not affect PTH receptor binding but significantly augmented a cAMP responsiveness to 10^?5 M forskolin and 1 ug/ml cholera toxin. Pertussis toxin (0.5 ug/ml) did not affect the PMA-induced augmentation of the PTH-stimulated cAMP production. PTH caused a complete homologous desensitization of [Ca²⁺]i response within 30 min. Pretreatment with 10^?4 M dibutyryl cAMP for 30 min and 6 h significantly reduced and completely blocked the PTH-induced increase in [Ca²⁺]i, respectively. Pretreatment with 10^?4 M Sp-cAMPS, a direct PKA activator, for 30 min completely blocked the PTH-induced increase in [Ca²⁺]i. Rp-cAMPS (10^?4 M), an antagonist of PKA, slightly but significantly antagonized the PTH-induced homologous desensitization of [Ca²⁺]i response. The present study indicates that the time of exposure to PKC activation is a critical determinant in modulating the cAMP system, while PKA activation counterregulatorily acts on the [Ca²⁺]i system, and that PKA activation is linked to the PTH-induced homologous desensitization of [Ca²⁺]i response. © 1994 Wiley-Liss, Inc.     

Protein kinase C-activated calcium channel in the osteoblast-like clonal osteosarcoma cell line UMR-106

The effects of protein kinase C stimulation on free cytosolic Ca2+ [( Ca2+]i) were studied in Fura 2-loaded UMR-106 cells. Stimulation of the protein kinase C with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol 13-acetate (TPA) and phorbol 12,13-diacetate or 1-oleoyl-2-acetylglycerol was followed by an increase in [Ca2+]i. The protein kinase C-induced increase in [Ca2+]i has a lag period, the duration of which was dependent on the stimulant and medium Ca2+ concentrations. With 2 microM TPA, the rise in [Ca2+]i peaked within 1.5 min, after which [Ca2+]i returned partially toward base line. The increase in [Ca2+]i was absolutely dependent on the presence of medium Ca2+ and was inhibited by the Ca2+ channel blockers nicardipine and verapamil. Cell stimulation also results in Ca2+ release from intracellular pool(s) which appears to be mediated by a Ca2+-dependent Ca2+ release mechanism. The reduction in [Ca2+]i was due to channel inactivation. Pretreatment of the cells with 1 nM TPA, 2 units/ml parathyroid hormone (PTH), or 15 microM forskolin blocked the effect of 2 microM TPA on [Ca2+]i. TPA and PTH were more potent inhibitors than was forskolin. The properties of this channel are compared to the cAMP-independent PTH-stimulated Ca2+ channel present in these cells.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.