Calcium oscillation induced by bradykinin in polyoma middle T antigen-transformed NIH3T3 fibroblasts: evidence for dependence on protein kinase C

Bradykinin (BK) triggered long lasting intracellular free calcium ([Ca2+]i) oscillation in polyoma middle T-transformed cell line MT3 cells but not in the parental NIH3T3 cells. This periodic [Ca2+]i fluctuation was extracellular Ca(2+)-dependent and blocked by pretreatments with Ca2+ channel blockers, SK&F 96365 or CdCl2, suggesting a crucial role of Ca2+ entry across the plasma membrane possibly through a receptor-operated Ca2+ channel. Brief pretreatment with phorbol myristate acetate (PMA) completely abolished the BK-induced [Ca2+]i oscillation, and a protein kinase C (PKC) inhibitor, H-7, reversed the effect of PMA, indicating involvement of PKC. On the other hand, in some cells, oscillatory changes in [Ca2+]i were seen without agonist stimulation. The spontaneous oscillation was also dependent on extracellular Ca2+, but neither treatment with PMA nor H-7 had any effect under the same conditions.     

Inhibition of bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilisation by phorbol ester in rat cultured vascular smooth muscle cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 microM) for 30 min almost abolished the BK-induced IP formation and Ca2+ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC50) and maximal inhibition of BK induced an increase in [Ca2+]i, were 7.8 +/- 0.3 M and 1 microM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 microM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca2+ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 microM PMA for either 1 or 24 h did not significantly change the K(D) and Bmax of the BK receptor for binding (control: K(D) = 1.7 +/- 0.2 nM; Bmax = 47.3 +/- 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, and zeta induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca2+ mobilisation in VSMCs.     

Inhibition of Bradykinin-Induced Phosphoinositide Hydrolysis and Ca Mobilisation by Phorbol Ester in Rat Cultured Vascular Smooth Muscle Cells

Regulation of the increase in inositol phosphate (IP) production and intracellular Ca²⁺ concentration ([Ca²⁺]_i by protein kinase C (PKC) was investigated in cultured rat vascular smooth muscle cells (VSMCs). Pretreatment of VSMCs with phorbol 12-myristate 14-acetate (PMA, 1 μM) for 30 min almost abolished the BK-induced IP formation and Ca²⁺ mobilisation. This inhibition was reduced after incubating the cells with PMA for 4 h, and within 24 h the BK-induced responses were greater than those of control cells. The concentrations of PMA giving a half-maximal (pEC₅₀) and maximal inhibition of BK induced an increase in [Ca²⁺]_i, were 7.8 ± 0.3 M and 1 μM, n = 8, respectively. Prior treatment of VSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate BK-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Paralleling the effect of PMA on the BK-induced IP formation and Ca²⁺ mobilisation, the translocation and downregulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of the cells with PMA for various times, translocation of PKC-, βI, βII, δ, ε, and ζ isozymes from the cytosol to the membrane were seen after 5 min, 30 min, 2 h, and 4 h of treatment. However, 24-h treatment caused a partial downregulation of these PKC isozymes in both fractions. Treatment of VSMCs with 1 μM PMA for either 1 or 24 h did not significantly change the K_D and B_max of the BK receptor for binding (control: K_D = 1.7 ± 0.2 nM; B_max = 47.3 ± 4.4 fmol/mg protein), indicating that BK receptors are not a site for the inhibitory effect of PMA on BK-induced responses. In conclusion, these resuts demonstrate that translocation of PKC-, βI, βII, δ, ε, and ζ induced by PMA caused an attenuation of BK-induced IPs accumulation and Ca²⁺ mobilisation in VSMCs.     

Regulation of 5-hydroxytryptamine-induced signal transduction in canine cultured aorta smooth muscle cells by phorbol ester.

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT-induced IPs formation and Ca2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-alpha, betaI, betaII, delta, epsilon, theta, and zeta induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca2+ mobilization in ASMCs.     

Stimulatory effect of prostaglandin F(2alpha) on Na-dependent phosphate transport in osteoblast-like cells

Prostaglandin F(2alpha) (PGF(2alpha)) has been reported to activate protein kinase C (PKC) through both phospholipase (PL) C and D, resulting in the proliferation of osteoblast-like cells. In addition, it has also been reported that Erk mitogen-activated protein kinase is also involved in the mechanism of PGF(2alpha)-induced proliferation of these cells. Recently, we have reported that several growth factors stimulate Na-dependent phosphate transport (Pi transport) activity of osteoblast-like cells, which has been recognized to play an important role in their mineralization. In the present study, we investigated the effect of PGF(2alpha) on Pi transport in MC3T3-E1 osteoblast-like cells. PGF(2alpha) stimulated Na-dependent Pi transport dose dependently in the range between 1nM and 10 micro M in MC3T3-E1 cells. The effect was time dependent up to 24h. Kinetic analysis revealed that PGF(2alpha) induces newly synthesized Pi transporter. Pretreatment with actinomycin D and cycloheximide suppressed PGF(2alpha)-induced enhancement of Pi transport. Combined effect of PMA and PGF(2alpha) was not additive in Pi transport. Calphostin C, a PKC inhibitor, dose-dependently suppressed Pi transport induced by PGF(2alpha). On the contrary, U0126, which inhibits an upstream kinase of Erk (MEK), did not affect PGF(2alpha)-induced enhancement of Pi transport. In conclusion, PGF(2alpha) stimulates Pi transport through activation of PKC in osteoblast-like cells.     

IL-6 is produced by osteoblasts and induces bone resorption

To examine the possible involvement of IL-6 in bone metabolism, a mouse osteoblastic cell line (MC3T3-E1) and primary osteoblast-like cells from fetal mouse calvaria were cultured with several systemic and local bone-resorbing agents and their expression of IL-6 mRNA was determined. Local bone-resorbing agents such as IL-1 alpha, IL-1 beta, TNF-alpha, and LPS greatly induced IL-6 mRNA expression in both MC3T3-E1 cells and primary osteoblast-like cells. Parathyroid hormone slightly increased expression of IL-6 mRNA in primary osteoblast-like cells but not in MC3T3-E1 cells. Neither IL-6 nor 1 alpha,25-dihydroxyvitamin D3 increased expression of IL-6 mRNA in either of the osteoblast-like cells. In agreement with the expression of IL-6 mRNA, biologically active IL-6 was produced in response to the treatment with IL-1 alpha, TNF-alpha, and LPS in MC3T3-E1 cells. Adding IL-6 dose dependently stimulated the release of 45Ca from prelabeled fetal mouse calvaria. Simultaneously adding suboptimal concentrations of IL-6 and IL-1 alpha induced bone resorption cooperatively. In accord with the increase in the release of 45Ca by IL-6, there were three times as many osteoclasts in the bone sections of calvaria cultured with IL-6 for 5 days as in the controls. IL-6 slightly suppressed alkaline phosphatase activity and collagen synthesis in MC3T3-E1 cells. These results indicate that IL-6 is also produced by osteoblasts, preferentially in response to local bone-resorbing agents, and it induces bone resorption both alone and in concert with other bone-resorbing agents.     

Cell density and growth-dependent down-regulation of both intracellular calcium responses to agonist stimuli and expression of smooth-surfaced endoplasmic reticulum in MC3T3-E1 osteoblast-like cells

A two-dimensional intracellular Ca(2+) ([Ca(2+)](i)) imaging system was used to examine the relationship between [Ca(2+)](i) handling and the proliferation of MC3T3-E1 osteoblast-like cells. The resting [Ca(2+)](i) level in densely cultured cells was 1.5 times higher than the [Ca(2+)](i) level in sparsely cultured cells or in other cell types (mouse fibroblasts, rat vascular smooth muscle cells, and bovine endothelial cells). A high resting [Ca(2+)](i) level may be specific for MC3T3-E1 cells. MC3T3-E1 cells were stimulated with ATP (10 microM), caffeine (10 mM), thapsigargin (1 microM), or ionomycin (10 microM), and the effect on the [Ca(2+)](i) level of MC3T3-E1 cells was studied. The percentage of responding cells and the degree of [Ca(2+)](i) elevation were high in the sparsely cultured cells and low in densely cultured cells. The rank order for the percentage of responding cells and magnitude of the Ca(2+) response to the stimuli was ionomycin > thapsigargin = ATP > caffeine and suggests the existence of differences among the various [Ca(2+)](i) channels. All Ca(2+) responses in the sparsely cultured MC3T3-E1 cells, unlike in other cell types, disappeared after the cells reached confluence. Heptanol treatment of densely cultured cells restored the Ca(2+) response, suggesting that cell-cell contact is involved with the confluence-dependent disappearance of the Ca(2+) response. Immunohistological analysis of type 1 inositol trisphosphate receptors and electron microscopy showed distinct expression of inositol trisphosphate receptor proteins and smooth-surfaced endoplasmic reticulum in sparsely cultured cells but reduced levels in densely cultured cells. These results indicate that the underlying basis of confluence-dependent [Ca(2+)](i) regulation is down-regulation of smooth-surfaced endoplasmic reticulum by cell-cell contacts.     

Prostaglandin D2 induces interleukin-6 synthesis via Ca2+ mobilization in osteoblasts: regulation by protein kinase C

We previously showed that prostaglandin (PG) D2 stimulates Ca2+ influx from extracellular space and activates phosphoinositidic (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D independently from PGE2 or PGF2alpha in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of PGD2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGD2 significantly stimulated IL-6 synthesis dose-dependently in the range between 10 nM and 10 microM. The depletion of extracellular Ca2+ by EGTA reduced the PGD2-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, significantly inhibited the PGD2-induced IL-6 synthesis. On the other hand, calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the synthesis of IL-6 induced by PGD2. In addition, U-73122, an inhibitor of phospholipase C, and propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the PGD2-induced IL-6 synthesis. These results strongly suggest that PGD2 stimulates IL-6 synthesis through intracellular Ca2+ mobilization in osteoblasts, and that the PKC activation by PGD2 itself regulates the over-synthesis of IL-6.     

Autoregulation of prostaglandin E2-induced Ca2+ influx in osteoblast-like cells: inhibition by self-induced activation of protein kinase C.

In cloned osteoblast-like MC3T3-E1 cells, prostaglandin E2 (PGE2) stimulated 45Ca2+ influx even in the presence of nifedipine, a Ca2+ antagonist that inhibits voltage-dependent Ca2+ channel, in a dose-dependent manner, attaining a maximum at 0.5 microM. Dose of PGE2 above 0.5 microM caused less than maximal stimulation. While PGE2 stimulated the formation of inositol trisphosphate dose dependently in the range between 1 nM and 10 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, which by itself had little effect on 45Ca2+ influx, significantly suppressed the 45Ca2+ influx induced by PGE2 in a dose-dependent manner between 1 nM and 1 microM. 4 alpha-Phorbol 12,13-didecanoate, a phorbol ester which is inactive for PKC, showed little effect in this capacity. Staurosporine, a PKC inhibitor, enhanced the PGE2-induced 45Ca2+ influx. On the other hand, dibutyryl cAMP had little effect on the 45Ca2+ influx induced by PGE2. Our data suggest that PGE2 regulates Ca2+ influx through self-induced activation of PKC. These results indicate that there is an autoregulatory mechanism in signal transduction by PGE2, and PGE2 modulates osteoblast functions through the interaction between Ca2+ influx and phosphoinositide hydrolysis in osteoblast-like cells.     

Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters

In this study, we examined the effects of T cell activators on the regulation of protein kinase C (PKC) isozymes present in thymocytes. Using affinity-purified anti-PKC antisera, we determined that the major PKC isoforms in murine thymocytes are PKC beta and PKC epsilon. The CD4+/CD8+ thymocyte subset expressed high levels of both PKC beta and PKC epsilon, whereas the CD4-/CD8- subset expressed much less of both. PKC beta was down-regulated following treatment of thymocytes with phorbol 12-myristate acetate (PMA) (2 x 10(-8) M) or ionomycin (0.4 microM). In contrast, PMA did not induce the down-regulation of PKC epsilon. Ionomycin alone, however, induced PKC epsilon down-regulation, similar to its effect on PKC beta. Similar observations were made on a promonocytic cell line, U937, which expresses PKC alpha, PKC beta (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Tsou, A.-P. (1989) Biochemistry 28, 3569-3576), and PKC epsilon. To facilitate the study of PKC beta and PKC epsilon, we established a Chinese hamster ovary cell line which expresses murine PKC epsilon in addition to endogenous PKC alpha and PKC beta. Both PKC isoforms (beta and epsilon) were mostly in particulate form. PMA treatment left the majority of immunoreactive PKC epsilon intact. By contrast, thrombin treatment caused the disappearance of particulate and cytosolic PKC epsilon (60% by 10 min and 80% by 1 h). PMA and thrombin promoted the down-regulation of PKC beta with similar kinetics (100% down-regulation by 3 h). These results indicate that: 1) thymocytes express PKC epsilon; and 2) this isozyme exhibits a novel form of regulation distinct from the other PKC isozymes.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

Potentiation of stimulus-induced insulin secretion in protein kinase C-deficient RINm5F cells.

The role of protein kinase C (PKC) in stimulus recognition and insulin secretion was investigated after long-term (24 h) treatment of RINm5F cells with phorbol 12-myristate 13-acetate (PMA). Three methods revealed that PKC was no longer detectable, and PMA-induced insulin secretion was abolished. Such PKC-deficient cells displayed enhanced insulin secretion (2-6-fold) in response to vasopressin and carbachol (activating phospholipase C) as well as to D-glyceraldehyde and alanine (promoting membrane depolarization and voltage-gated Ca2+ influx). Insulin release stimulated by 1-oleoyl-2-acetylglycerol (OAG) was also greater in PKC-deficient cells. OAG caused membrane depolarization and raised the cytosolic Ca2+ concentration ([Ca2+]i), both of which were unaffected by PKC down-regulation. Except for that caused by vasopressin, the secretagogue-induced [Ca2+]i elevations were similar in control and PKC-depleted cells. The [Ca2+]i rise evoked by vasopressin was enhanced during the early phase (observed both in cell suspensions and at the single cell level) and the stimulation of diacylglycerol production was also augmented. These findings suggest more efficient activation of phospholipase C by vasopressin after PKC depletion. Electrically permeabilized cells were used to test whether the release process is facilitated after long-term PMA treatment. PKC deficiency was associated with only slightly increased responsiveness to half-maximally (2 microM) but not to maximally stimulatory Ca2+ concentrations. At 2 microM-Ca2+ vasopressin caused secretion, which was also augmented by PMA pretreatment. The difference between intact and permeabilized cells could indicate the loss in the latter of soluble factors which mediate the enhanced secretory responses. However, changes in cyclic AMP production could not explain the difference. These results demonstrate that PKC not only exerts inhibitory influences on the coupling of receptors to phospholipase C but also interferes with more distal steps implicated in insulin secretion.     

Inhibitory effect of 17 beta -estradiol on prostaglandin E2-induced phosphoinositide hydrolysis in osteoblast-like cells.

We examined the effect of estradiol on PGE2-induced phosphoinositide hydrolysis and cAMP production in cloned osteoblast-like MC3T3-E1 cells. 17 beta -Estradiol pretreatment significantly inhibited the formation of inositol phosphates induced by 10 microM PGE2 in a dose-dependent manner between 1 pM and 10 nM. This effect of 17 beta -estradiol was dependent on the time of pretreatment and submaximum inhibition was observed at 4 h. However, 17 beta -estradiol had little effect on the formation of inositol phosphates induced by 20 mM NaF, a GTP-binding protein activator. The cAMP production induced by PGE2 was not influenced by 17 beta -estradiol. These results suggest that 17 beta -estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-E1 cells.     

Protein kinase C activation by interleukin (IL)-1 limits IL-1-induced IL-6 synthesis in osteoblast-like cells: Involvement of phosphatidylcholine-specific phospholipase C

We investigated the regulatory mechanism of interleukin-6 (IL-6) synthesis induced by interleukin-1 (IL-1) in osteoblast-like MC3T3-E1 cells. IL-1 stimulated the secretion of IL-6 in a dose-dependent manner in the range between 0.1 and 100 ng/ml. Staurosporine and calphostin C, inhibitors of protein kinase C (PKC), significantly enhanced the IL-1-induced secretion of IL-6. The stimulative effect of IL-1 was markedly amplified in PKC down-regulated MC3T3-E1 cells. IL-1 produced diacylglycerol in MC3T3-E1 cells. IL-1 had little effect on the formation of inositol phosphates and choline. On the contrary, IL-1 significantly stimulated the formation of phosphocholine dose-dependently. D-609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed the IL-1-induced diacylglycerol production. The IL-1-induced IL-6 secretion was significantly enhanced by D-609. These results indicate that IL-1 activates PKC via phosphatidylcholine-specific phospholipase C in osteoblast-like cells, and the PKC activation then limits IL-6 synthesis induced by IL-1 itself. J. Cell. Biochem. 67:103–111, 1997. © 1997 Wiley-Liss, Inc.     

Involvement of p38 mitogen-activated protein kinase in basic fibroblast growth factor-induced interleukin-6 synthesis in osteoblasts.

We previously showed that basic fibroblast growth factor (bFGF)-induced activation of protein kinase C (PKC) via phosphatidylinositol-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D suppresses interleukin-6 (IL-6) synthesis by bFGF itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism underlying the bFGF-induced IL-6 synthesis in MC3T3-E1 cells. bFGF time-dependently induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase. SB203580, a specific inhibitor of p38 MAP kinase, suppressed the bFGF-induced IL-6 synthesis dose-dependently. The phosphorylation of p38 MAP kinase by bFGF was suppressed by TMB-8, an inhibitor of intracellular Ca(2+) mobilization, or the depletion of extracellular Ca(2+) with EGTA. A23187, a Ca-ionophore, stimulated the phosphorylation of p38 MAP kinase. SB203580 inhibited the A23187-induced synthesis of IL-6. 1-Oleoyl-2-acetyl-sn-glycerol, a synthetic diacylglycerol activating PKC, reduced the bFGF-induced IL-6 synthesis. 12-O-Tetradecanoylphorbol-13-acetate, an activator of PKC, attenuated the phosphorylation of p38 MAP kinase by bFGF, but did not affect the A23187-induced phosphorylation. These results strongly suggest that bFGF-induced IL-6 synthesis is mediated via p38 MAP kinase activation in osteoblasts, and that PKC acts at a point upstream from p38 MAP kinase.     

Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin expression, and mineralization in MC3T3-E1 cells. Inhibition of AA uptake by SVCT2 short isoform functioning as a dominant-negative mutant not only robustly attenuated PGE2-induced markers expression and mineralization, but also decreased their basal levels. However, upregulation of AA uptake resulted from PGE2-induced plasma membrane translocation of cytoplasm SVCT2, and this effect was abolished by pretreatment with EP4 receptor antagonist, AH-23848B or cAMP-dependent protein kinase A (PKA) inhibitor, H-89. Moreover, we showed SVCT2 physically interacted with PKA in immunoprecipitates, and PKA phosphorylated SVCT2 in vitro and in intact cells at Ser402 and Ser639 sites; however, mutation of Ser402 or/and Ser639 in SVCT2 severely diminished SVCT2 translocation in response to PGE2. Together, these results suggest that PGE2-induced SVCT2 plasma membrane translocation through EP4 receptor and subsequent phosphorylation of SVCT2 at Ser402 and Ser639 sites by PKA results in an increase of AA uptake and consequent promotion of osteoblast-like differentiation in MC3T3-E1 cells.     

Phorbol ester and endothelin-1 alter functional expression of Na+/Ca2+ exchange, K+, and Ca2+ currents in cultured neonatal rat myocytes

Endothelin-1 (ET-1) and activation of protein kinase C (PKC) have been implicated in alterations of myocyte function in cardiac hypertrophy and heart failure. Changes in cellular Ca2+ handling and electrophysiological properties also occur in these states and may contribute to mechanical dysfunction and arrhythmias. While ET-1 or PKC stimulation induces cellular hypertrophy in cultured neonatal rat ventricular myocytes (NRVMs), a system widely used in studies of hypertrophic signaling, there is little data about electrophysiological changes. Here we studied the effects of ET-1 (100 nM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 μM) on ionic currents in NRVMs. The acute effects of PMA or ET-1 (≤30 min) were small or insignificant. However, PMA or ET-1 exposure for 48-72 h increased cell capacitance by 100 or 25%, respectively, indicating cellular hypertrophy. ET-1 also slightly increased Ca2+ current density (T and L type). Na+/Ca2+ exchange current was increased by chronic pretreatment with either PMA or ET-1. In contrast, transient outward and delayed rectifier K+ currents were strongly downregulated by PMA or ET-1 pretreatment. Inward rectifier K+ current tended toward a decrease at larger negative potential, but time-independent outward K+ current was unaltered by either treatment. The enhanced inward and reduced outward currents also result in action potential prolongation after PMA or ET-1 pretreatment. We conclude that chronic PMA or ET-1 exposure in cultured NRVMs causes altered functional expression of cardiac ion currents, which mimic electrophysiological changes seen in whole animal and human hypertrophy and heart failure.