Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The osteoclastogenic factor of osteoblastic origin has recently been elucidated as a novel Tumor Necrosis Factor (TNF)-ligand family member, termed osteoclast differentiation factor (ODF). Using a semiquantitative RT-PCR approach, we sought to determine the mRNA expression of ODF and its decoy receptor, osteoprotegerin (OPG), in a selection of osteoblastic cell lines and in response to three factors representative of different signal transduction pathways, vitamin D receptor, protein kinase A or gp130. Each osteotropic agent, either 1,25-(OH)2D3, PTH or IL-11, promoted an increase in the ratio of ODF:OPG, with maximal stimulation occurring at 24 h, 4 h, and 8 h, respectively, and furthermore each was shown to act in a dose-dependent manner. This report establishes that osteoblastic cell lines incapable of supporting osteoclast formation have markedly reduced ODF expression and also illustrates the importance of the relative abundance of ODF compared with the levels of OPG for the induction of osteoclastogenesis.
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PMID:Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. 979 88

PTH is an 84-amino acid protein. Occupancy of its cognate receptor generally results in activation of adenylyl cyclase and/or phosphoinositide-specific phospholipase Cbeta (PLCbeta). In the kidney, PTH receptors are present on proximal and distal tubule cells. In proximal tubules, PTH induces calcium signaling, typified by a transient rise in intracellular calcium ([Ca2+]i) and inositol trisphosphate formation, but does not affect calcium absorption. By contrast, in distal tubules, PTH increases calcium absorption that is associated with a slow and sustained rise in [Ca2+]i, but does not stimulate phospholipase C (PLC) or cause inositol trisphosphate accumulation. Nonetheless, stimulation of distal calcium transport requires activation of protein kinase C (PKC) and protein kinase A. We now characterize the origin of the differential effects of ligand occupancy by using synthetic human PTH analogs that preferentially activate adenylyl cyclase and/or PLCbeta. We further tested the hypothesis that phospholipase D is responsible for PKC activation in distal tubule cells. PTH-(1-31) increased [Ca2+]i in distal tubule but not in proximal tubule cells, whereas PTH-(3-34) caused a partial increase in [Ca2+]i in proximal cells, but had no effect in distal cells. PTH-(7-34) blocked increases in [Ca2+]i in distal tubule cells stimulated by PTH-(1-34) and PTH-(1-31). The PLC inhibitor U73122 abolished the PTH-induced rise in [Ca2+]i and inositol trisphosphate formation by proximal tubule cells, but had no effect on PTH-stimulated Ca2+ uptake by distal tubule cells. These results support the view that activation of PKC by PTH in distal tubule cells does not involve PLCbeta. PTH did, however, activate phospholipase D with attendant formation of diacylglycerol in distal cells. As activation of PKC is required for induction of calcium transport by PTH, we conclude that PTH receptors are capable of activating multiple phospholipases and that the structural requirements for such activation differ in proximal and distal tubule cells.
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PMID:Cell-specific signaling and structure-activity relations of parathyroid hormone analogs in mouse kidney cells. 988 39

PTH induces c-fos expression rapidly and transiently in osteoblastic cells and requires the activity of the cAMP response element-binding protein (CREB). Here we provide evidence that protein kinase A (PKA) is the enzyme responsible for phosphorylating CREB at serine 133 (S133) and that this event is required for PTH-induced c-fos expression. PTH increases the level of phosphorylation of CREB at S133 in a time- and dose-dependent manner, correlating with the time and level of activation of PKA in response to PTH. PTH-(1-34) and -(1-31), each known to activate the cAMP pathway, induced the phosphorylation of CREB and increased the levels of c-fos messenger RNA, whereas PTH-(3-34), -(13-34), and -(28-48) could not. Specific inhibitors of calcium/calmodulin-dependent protein kinases and protein kinase C could not inhibit CREB phosphorylation or c-fos expression in response to PTH; however, H-89, a specific inhibitor of PKA, could do so in a dose-dependent manner. In addition, PTH-induced c-fos promoter activity was completely inhibited in a dose-dependent fashion by transfection of the heat-stable inhibitor of PKA. Taken together, these data provide strong evidence that PKA is the enzyme responsible for phosphorylating CREB at S133 in response to PTH and that PKA activity is required for PTH-induced c-fos expression.
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PMID:Increased osteoblastic c-fos expression by parathyroid hormone requires protein kinase A phosphorylation of the cyclic adenosine 3',5'-monophosphate response element-binding protein at serine 133. 1006 51

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.
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PMID:Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. 1006 56

Reflecting the prime role of 1alpha,25(OH)2D3 in calcium homeostasis, the activity of 25-hydroxyvitamin D3 1alpha-hydroxylase, a key enzyme for 1alpha,25(OH)2D3 biosynthesis, is tightly regulated by 1alpha,25(OH)2D3, PTH and calcitonin. Its significant activity is found in kidney, though the enzymatic activity is also reported in extra-renal tissues. In the present study, we found that the 1alpha-hydroxylase gene abundantly expresses in kidney, and at low levels in other tissues and in some cell lines. Positive and negative regulations of 1alpha-hydroxylase gene by PTH, calcitonin, or 1alpha,25(OH)2D3 were observed at transcriptional levels in kidneys of animals and in a mouse proximal tubule cell line. Moreover, the protein kinase A inhibitor abrogated the PTH-mediated positive regulation. In mice lacking the vitamin D receptor, the 1alpha-hydroxylase gene expression was overinduced, and the inducible effect of either PTH or calcitonin, but not the repression by 1alpha,25(OH)2D3, was evident. Thus, vitamin D receptor is essential for the negative regulation by 1alpha,25(OH)2D3. Moreover, we demonstrate that renal 1alpha-hydroxylase gene expression in chronic renal failure model rats was decreased and the positive effect by PTH and calcitonin was diminished. The present study demonstrates that PTH and calcitonin positively regulate renal 1alpha-hydroxylase gene expression via PKA-dependent and independent pathway, respectively, and that 1alpha,25(OH)2D3 negatively regulates it mediated by vitamin D receptor. Furthermore, in a moderate state of chronic renal failure, renal cells expressing the 1alpha-hydroxylase gene appear to have diminished potential in response to PTH and calcitonin.
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PMID:Positive and negative regulations of the renal 25-hydroxyvitamin D3 1alpha-hydroxylase gene by parathyroid hormone, calcitonin, and 1alpha,25(OH)2D3 in intact animals. 1021 75

PTH has anabolic and catabolic effects in bone through activation of the PTH-1 (PTH/PTHrP) receptor and the cAMP/protein kinase A pathway. The effects of agents that regulate cAMP in nontransformed osteoblasts in relation to cell differentiation have not been described. The purpose of this study was to determine the effects of PTH fragments with differing cAMP-stimulating activity, and nonPTH cAMP regulators on PTH-1 receptor expression and activity, and osteoblast differentiation in vitro using MC3T3-E1 and primary rat calvarial cells. PTH (1-34), but not PTH (53-84), (7-34), or PTHrP (107-139) treatment (24 h) resulted in down-regulation of steady-state messenger RNA for the PTH-1 receptor. Forskolin (a stimulator of cAMP accumulation) also down regulated the PTH-1 receptor, whereas 9-(tetrahydro-2-furyl) adenine (THFA) (an inhibitor of adenylyl cyclase) had no effect. Similarly, PTH (1-34) treatment for 48 h abolished PTHrP binding to cell surface receptors; however, neither the PTH analogs nor the cAMP regulating agents altered PTH binding or numbers of binding sites on osteoblastic cells. Basal levels of cAMP were reduced in cultured cells treated for 6 days with PTH (7-34) or THFA compared with controls. In contrast, PTH-stimulated cAMP levels were significantly increased in cultures treated with PTH (7-34) and THFA for 6 days during osteoblast differentiation and were decreased in cultures treated with PTH (1-34) and forskolin compared with controls. To evaluate effects of the cAMP pathway on osteoblast differentiation, cultures were treated continuously with PTH analogs and cAMP regulators during an 18-day differentiation regime, total RNA was isolated at multiple time points, and Northern blot analysis for osteocalcin (OCN) was performed. THFA and PTH (7-34)-treated cultures had increased OCN expression; whereas, PTH (1-34) and forskolin reduced OCN expression. Interestingly, PTH (7-34) and THFA-treated cultures had increased mineralized nodule formation, in contrast to PTH (1-34) and forskolin treatment, which reduced nodule formation. Similarly, calcium accumulation in cultures was significantly increased in the PTH (7-34) and THFA-treated cultures and reduced in the PTH (1-34) and forskolin-treated cultures. These data demonstrate that agents that increase cAMP down regulate PTH-1 receptor messenger RNA and inhibit osteoblast differentiation in vitro. Agents that reduce or block adenylyl cyclase or cAMP activity do not alter PTH-1 receptor expression or binding, but have striking effects on promoting osteoblast differentiation. We conclude that many effects of PTH on osteoblasts may be mimicked or antagonized by agents that alter cAMP activity and bypass the PTH-1 receptor.
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PMID:3',5'-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro. 1038 9

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH(2), which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH(2) to stimulate bone growth despite its having hPTH-(1-31)NH(2)'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH(2)'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2-10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10-50 nmol of hPTH-(1-30)NH(2)/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH(2)/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH(2), [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-31)NH(2), hPTH-(1-34)NH(2) and [Leu(27)]cyclo(Glu(22)-Lys(26))-hPTH-(1-34)NH(2). These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause.
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PMID:Stimulation of femoral trabecular bone growth in ovariectomized rats by human parathyroid hormone (hPTH)-(1-30)NH(2). 1043 Jun 48

Many G protein-coupled receptor agonists activate p42/p44 mitogen-activated protein kinase (MAPK), using signaling pathways that are a function of receptor, G protein-coupled, and effector complement. In opossum kidney (OK) cells, activation of endogenous PTH receptors caused a time- (peak within 15-30 min, sustained for approximately 2 h) and dose-dependent (EC50 approximately 3 x 10(-10) M) activation of MAPK. Immunoblot analysis with an activation- specific MAPK antibody indicated that PTH activated both p42 and p44 MAPK. Epidermal growth factor (EGF) also activated p42 and p44MAPK in a time- (peak at 5 min, return to basal within 2 h) and dose-dependent (EC50 approximately 3 ng/ml) fashion. PTH-dependent MAPK activation was mimicked by the protein kinase C activator (PKC) phorbol myristate acetate (PMA), and the protein kinase A activators 8 bromo-cAMP (8-Br-cAMP) and forskolin but was not affected by pertussis toxin pretreatment. PMA or 8-Br-cAMP pretreatment blocked MAPK activation by reexposure to each kinase activator but caused no significant reduction in MAPK activation by PTH. MAPK activation by PTH, EGF, and 8-Br-cAMP was inhibited by the MAPK kinase inhibitor PD98059 and an EGF receptor (EGFR)-selective inhibitor tyrphostin AG1478. AG1478 also blocked MAPK activation by insulin-like growth factor-1 and platelet-derived growth factor. EGF and PTH caused time- and AG1478-sensitive phosphorylation of the EGFR, but EGFR desensitization did not affect MAPK activation by PTH. EGF, PMA, and low doses of PTH (10(12) to 10(-9) M) stimulated while 8-Br-cAMP and high doses of PTH (10(-8) to 10(-6) M) inhibited [3H]thymidine uptake. These data demonstrate that PTH activates MAPK and suggest that PKC, protein kinase A, and the EGFR play roles in PTH signaling. The biphasic effect of PTH on DNA synthesis suggests that MAPK activation by the hormone leads to distinct cellular responses.
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PMID:Parathyroid hormone activates mitogen-activated protein kinase in opossum kidney cells. 1057 43

To examine the importance of the N- or C-termini of PTH(1-34) the effects of truncated fragments of PTH on human receptors in osteoblast-like SaOS-2 cells and rat receptors in rats ROS 17/2 cells were examined. Fura-2-loaded cells were used to monitor cytosolic free Ca(2+) concentration ([Ca2+]i), and the Cytosensor microphysiometer was used to monitor extracellular acidification rate (ECAR). C-terminally truncated fragments (1-31) and (1-28) of hPTH(1-34)NH(2) stimulated an increase in [Ca(2+)](i) and ECAR in both cell lines. hPTH(3-34)NH(2) and other N-terminally truncated fragments did not stimulate [Ca(2+)](i) or ECAR in either cell type. The signal transduction pathway of PTH-induced ECAR in ROS 17/2 cells was investigated to compare with previous results in SaOS-2 cells. Potentiation by IBMX, attenuation by 8Br-cAMP and lack of effect of the PKC inhibitor chelerythrine chloride support a cAMP/PKA-mediated signal transduction pathway in ROS 17/2, while the protein kinase C pathway was predominant in SaOS-2 cells. We conclude that the intact N-terminus of PTH is essential in PTH signaling mediated via either the cAMP/PKA or inositol lipid/Ca(2+)/PKC pathways in osteoblast-like cells.
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PMID:Ca(2+) and extracellular acidification rate responses to parathyroid hormone fragments in rat ROS 17/2 and human SaOS-2 cells. 1060 May 23

Parathyroid hormone (PTH-(1-34)) potently suppresses apatite deposition in osteoblastic cultures. These inhibitory effects are mediated through signaling events following PTH receptor binding. Using both selective inhibitors and activators of protein kinase A (PKA), this study shows that a transient activation of PKA is sufficient to account for PTH's inhibition of apatite deposition. This inhibition is not a result of reduced cell proliferation, reduced alkaline phosphatase activity, increased collagenase production, or lowering medium pH. Rather, data suggest a functional relationship between matrix assembly and apatite deposition in vitro. Bone sialoprotein (BSP) and apatite co-localize in the extracellular matrix of mineralizing cultures, with matrix deposition of BSP temporally preceding that of apatite. Transient activation of PKA by either PTH-(1-34) or short term cAMP analog treatment blocks the deposition of BSP in the extracellular matrix without a significant reduction in the total amount of BSP synthesized and secreted. This effect is reversible after allowing the cultures to recover in the absence of PKA activators for several days. Thus, a transient activation of PKA may suppress mineral deposition in vitro as a consequence of altering the assembly of an extracellular matrix permissive for apatite formation.
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PMID:Reversible suppression of in vitro biomineralization by activation of protein kinase A. 1075 13


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