Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously shown that 24,25-(OH)2D3 plays a major role in resting zone (RC) chondrocyte differentiation and that this vitamin D metabolite regulates protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 24,25-(OH)2D3 to stimulate PKC activation. Confluent, fourth passage RC cells from rat costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of RC cultures with 24,25-(OH)2D3 for 90 min produced a dose-dependent increase in diacylglycerol (DAG). Addition of R59022, a diacylglycerol kinase inhibitor, significantly increased PKC activity in cultures treated with 24,25-(OH)2D3. Addition of dioctanoylglycerol (DOG) to plasma membranes isolated from RC increased PKC activity 447-fold. Addition of pertussis toxin or cholera toxin to control cultures elevated basal PKC activity. When added together with 10(-9) M 24,25-(OH)2D3, there was an additive effect on PKC activity but in cultures treated with 10(-8) M 24,25-(OH)2D3, only the hormone-dependent stimulation of PKC was observed. The phospholipase C inhibitor, U73-122, had no effect on PKC activity, indicating that the DAG produced in response to 24,25-(OH)2D3 is not derived from phosphatidylinositol. Addition of the tyrosine kinase inhibitor, genistein, also had no effect on 24,25-(OH)2D3-stimulated PKC, further supporting the hypothesis that phospholipase C is not involved in the mechanism and that phospholipase D is responsible for the increase in DAG production. Phospholipase A2 inhibitors, quinacrine and AACOCF3, and the cyclooxygenase inhibitor indomethacin increased PKC activity in the RC cultures. Exogenous PGE2, one of the downstream products of phospholipase A2 action, inhibited PKC activity. These results suggest that 24,25-(OH)2D3 regulates PKC activity by two distinct phospholipid-dependent mechanisms: production of DAG via phospholipase D and inhibition of the production of PGE2 via inhibition of phospholipase A2 and cyclooxygenase.
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PMID:24,25-(OH)2D3 regulates protein kinase C through two distinct phospholipid-dependent mechanisms. 895

The 5'-flanking region of the human phospholipase C-gamma1 gene was isolated from a human P1 genomic DNA library. The S1-nuclease mapping and primer extension analysis revealed that there is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-gamma1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The fragment +135 to -877 in the 5'-flanking region of the human phospholipase C-gamma1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity and responded to 1,25-(OH)2D3 in transiently transfected human keratinocytes. Deletion and mutation studies of the fragment +135 to -877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases (DR6), AGGTCAgaccacTGGACA, located between -786 and -803 base pairs. Incubation of the oligonucleotide containing the DR6 with keratinocyte nuclear extracts produced a specific protein-DNA complex that shifted to a higher molecular weight form upon the addition of an antibody specific to the 1,25-(OH)2D3 receptor. Therefore, the 5'-flanking region of the human phospholipase C-gamma1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by 1, 25-(OH)2D3.
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PMID:Cloning of the human phospholipase C-gamma1 promoter and identification of a DR6-type vitamin D-responsive element. 904 85

We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca2+]i) within 5 s by mobilizing calcium from the endoplasmic reticulum and the formation of inositol 1,4, 5-trisphosphate and diacylglycerol. Because the activation of effectors as phospholipase C (PLC) coupled to G-proteins is the early event in the signal transduction pathway leading to the inositol 1,4,5-trisphosphate formation and to [Ca2+]i increase, we described different PLC isoforms (beta1, beta2, gamma1, and gamma2, but not beta4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C beta was involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17beta estradiol, and PLC gamma was ineffective. The data also showed that only a PLC beta1 linked to a Pertussis toxin-insensitive G-protein and a PLC beta2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17beta estradiol on the mobilization of Ca2+ from intracellular Ca2+ stores. In conclusion, these results may be an important step toward understanding membrane effects of these steroids and may be an additional argument in favor of membrane receptors to steroid hormones.
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PMID:Phospholipase C beta and membrane action of calcitriol and estradiol. 911 51

Squamous cell carcinomas (SCC) derived from human epidermis fail to differentiate normally under the influence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] despite the presence of the vitamin D receptor. Previous studies from our laboratory showed that phospholipase C-gamma1 (PLC-gamma1) was upregulated transcriptionally by 1,25(OH)2D3 in normal human keratinocytes, and a vitamin D responsive element (VDRE) in its promoter region has been identified. To examine the inducibility of human PLC-gamma1 transcription by 1,25(OH)2D3 and/or retinoic acid in SCC cell lines, we transiently transfected SCC4 and SCC12B2 cells with human PLC-gamma1 promoter-luciferase constructs containing the VDRE and tested the response of these constructs to 1,25(OH)2D3 and/or all-trans retinoic acid. The induction of the human PLC-gamma1 VDRE by 1,25(OH)2D3 was synergistic with all-trans retinoic acid in normal human keratinocytes, but none of the constructs was induced by 1,25(OH)2D3 and/or all-trans retinoic acid in SCC4 and SCC12B2 cells. In contrast, the construct containing the VDRE of the human 24-hydroxylase gene was induced several fold by 1,25(OH)2D3 in normal human keratinocytes and by both 1,25(OH)2D3 and all-trans retinoic acid in SCC4 and SCC12B2 cells. DNA mobility shift assays showed that both the vitamin D receptor and the retinoic acid receptor in SCC4 and SCC12B2 cells bound the human PLC-gamma1 VDRE similarly to that seen in normal keratinocytes. The data indicate that the VDRE in the human PLC-gamma1 gene is not functional in SCC4 and SCC12B2 cells, unlike normal human keratinocytes, even though vitamin D receptors bind normally to it. Failure of transcriptional control of the PLC-gamma1 gene by 1,25(OH)2D3 suggests the lack of a cofactor(s) linking the VDRE to the transcriptional machinery.
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PMID:Differential regulation of vitamin D responsive elements in normal and transformed keratinocytes. 957 36

1,25-(OH)2D3 (1,25) exerts its effects on growth plate chondrocytes through classical vitamin D (VDR) receptor-dependent mechanisms, resulting in mineralization of the extracellular matrix. Recent studies have shown that membrane-mediated mechanisms are involved as well. 1,25 targets cells in the prehypertrophic and upper hypertrophic zones of the costochondral cartilage growth plate (GC cells), resulting in increased specific activity of alkaline phosphatase (ALP), phospholipase A2 (PLA2), and matrix metalloproteinases (MMPs). At the cellular level, 1,25 action results in rapid changes in arachidonic acid (AA) release and re-incorporation, alterations in membrane fluidity and Ca ion flux, and increased prostaglandin E1 and E2 (PGE2) production. Protein kinase C (PKC) is activated in a phospholipase C (PLC) dependent-mechanism, due in part to the increased production of diacylglycerol (DAG). In addition, AA acts directly on the cell to increase PKC specific activity. AA also provides a substrate for cyclooxygenase (COX), resulting in PGE2 production. 1,25 mediates its effects through COX-1, the constitutive enzyme, but not COX-2, the inducible enzyme. Time course studies using specific inhibitors of COX-1 show that AA stimulates PKC activity and PKC then stimulates PGE2 production. PGE2 acts as a mediator of 1,25 action on the cells, also stimulating PKC activity. The rapid effects of 1,25 on PKC are nongenomic, occurring within 3 min and reaching maximal activation by 9 min. It promotes translocation of PKC to the plasma membrane. When 1,25 is incubated directly with isolated plasma membranes, PKCalpha is stimulated although PKCzeta is also present. In contrast, when isolated matrix vesicles (MVs) are incubated with 1,25, PKCzeta is inhibited and PKCalpha is unaffected. These membrane-mediated effects are due to the presence of a specific membrane vitamin D receptor (mVDR) that is distinct from the classical cytosolic VDR. Studies using 1,25 analogs with reduced binding affinity for the classical VDR, confirm that rapid activation of PKC by 1,25 is not VDR dependent. The membrane-mediated effects of 1,25 are critical to the regulation of events in the extracellular matrix produced by the chondrocytes. MVs are extracellular organelles associated with maturation of the matrix, preparing it for mineralization. MV composition is under genomic control, involving VDR-mechanisms. In the matrix, no new gene expression or protein synthesis can occur, however. Differential distribution of PKC isoforms and their nongenomic regulation by 1,25 is one way for the chondrocyte to control events at sites distant from the cell. GC cells contain 1a-hydroxylase and produce 1,25; this production is regulated by 1,25, 24,25, and dexamethasone. 1,25 stimulates MMPs in the MVs, resulting in increased proteoglycan degradation in mineralization gels, and increased activation of latent transforming growth factor-beta 1 (TGF-beta1).
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PMID:1,25-(OH)2D3 modulates growth plate chondrocytes via membrane receptor-mediated protein kinase C by a mechanism that involves changes in phospholipid metabolism and the action of arachidonic acid and PGE2. 1032 81

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.
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PMID:Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes. 1038 91

Regulation of muscle cell Ca(2+) metabolism by 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] is mediated by the classic nuclear mechanism and a fast, nongenomic mode of action that activates signal transduction pathways. The role of individual protein kinase C (PKC) isoforms in the regulation of intracellular Ca(2+) levels ([Ca(2+)](i)) by the hormone was investigated in cultured proliferating (myoblasts) and differentiated (myotubes) chick skeletal muscle cells. 1,25(OH)(2)D(3) (10(-9) M) induced a rapid (30- to 60-s) and sustained (>5-min) increase in [Ca(2+)](i) which was markedly higher in myotubes than in myoblasts. The effect was suppressed by the PKC inhibitor calphostin C. In differentiated cells, PKC activity increased in the particulate fraction and decreased in cytosol to a greater extent than in proliferating cells after 5-min treatment with 1,25(OH)(2)D(3). By Western blot analysis, these changes were correlated to translocation of the PKC alpha isoform from cytosol to the particulate fraction, which was more pronounced in myotubes than in myoblasts. Specific inhibition of PKC alpha activity using antibodies against this isoform decreased the 1, 25(OH)(2)D(3)-induced [Ca(2+)](i) sustained response associated with Ca(2+) influx through voltage-dependent calcium channels. Neomycin, a phospholipase C (PLC) inhibitor, blocked its effects on [Ca(2+)](i), PKC activity, and translocation of PKC alpha. Exposure of myotubes to 1,2-dioleyl-rac-glycerol (1,2-diolein), also increased [Ca(2+)](i), PKC activity, and the amount of PKC alpha associated with the particulate fraction. Changes in [Ca(2+)](i) induced by diolein were inhibited by calphostin C and nifedipine. The results indicate that PKC alpha activation via PLC-catalyzed phosphoinositide hydrolysis is part of the mechanism by which 1, 25(OH)(2)D(3) regulates muscle intracellular Ca(2+) through modulation of the Ca(2+) influx pathway of the Ca(2+) response to the sterol.
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PMID:Role of protein kinase C in 1,25(OH)(2)-vitamin D(3) modulation of intracellular calcium during development of skeletal muscle cells in culture. 1072 87

Phospholipase C-gamma1 (PLC-gamma1) is the most abundant member of the phospholipase C family expressed in human keratinocytes. PLC-gamma1 is induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) in normal keratinocytes via a DR6-type vitamin D responsive element. This regulation is not observed in transformed keratinocytes. The role of PLC-gamma1 in mediating 1alpha,25(OH)(2)D(3) and calcium-regulated differentiation was then tested. Both specific PLC inhibitors and antisense constructs which selectively block PLC-gamma1 production prevented 1alpha,25(OH)(2)D(3) and calcium from inducing markers of differentiation such as involucrin and transglutaminase. These studies demonstrate that PLC-gamma1 induction by 1alpha,25(OH)(2)D(3) is critical to the ability of this hormone to regulate keratinocyte differentiation.
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PMID:The role of phospholipase C-gamma1 in 1alpha,25-dihydroxyvitamin D(3) regulated keratinocyte differentiation. 1117 42

In previous work we have demonstrated that the steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] stimulates in skeletal muscle cells the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2. In the present study we evaluated the involvement of Ca(2+) and protein kinase C (PKC) on 1,25(OH)(2)D(3)-induced activation of MAP kinase. The hormone response was found to depend on PKC stimulation since it was attenuated by the PKC inhibitors calphostin C (100 nM) and bisindolylmaleimide I (30 nM) and PKC downregulation by prolonged treatment with the phorbol ester TPA (1 microM). Removal of external Ca(2+), chelation of intracellular Ca(2+) with BAPTA (5 microM), inhibition of phosphoinositide-phospholipase C (PLC) by neomycin, the calmodulin antagonist fluphenazine (50 microM) and the specific inhibitor of calmodulin kinase II, KN-62 (10 microM), significantly decreased 1,25(OH)(2)D(3)-activation of MAP kinase. In addition, the Ca(2+)-channel blocker verapamil (5 microM) suppressed hormone-induced MAP kinase activity in these cells. Furthermore, the Ca(2+)-mobilizing agent thapsigargin and the Ca(2+)-inophore A23187 paralleled the phosphorylation of MAP kinase observed with 1,25(OH)(2)D(3). Taken together, these results indicate that PKC and Ca(2+) are two upstream activators mediating the effects of 1,25(OH)(2)D(3) on MAP kinase in skeletal muscle cells.
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PMID:The stimulation of MAP kinase by 1,25(OH)(2)-vitamin D(3) in skeletal muscle cells is mediated by protein kinase C and calcium. 1122 76

When osteoblasts are cultured on surfaces of increasing microroughness, they exhibit decreases in proliferation, increases in differentiation and local factor production, and enhanced response to 1alpha,25(OH)(2)D(3). The cells interact with surfaces through integrins, which signal by the same pathways used by 1alpha,25(OH)(2)D(3), including protein kinase C via phospholipase C and protein kinase A via phospholipase A(2). This provides opportunities for crosstalk that may contribute to the synergistic effects of surface roughness and the vitamin D metabolite. Because these pathways converge at mitogen-activated protein kinase (MAPK), we tested the hypothesis that the extracellular signal-regulated kinase (ERK1/2) subclass of MAPKs mediates the effects of surface roughness and 1alpha,25(OH)(2)D(3). MG63 osteoblast-like osteosarcoma cells were cultured on commercially pure Ti disks with various surface roughnesses: pretreatment (PT; 0.6 microm average roughness [Ra]), coarse grit-blasted and acid-etched (SLA; 4 microm RA), and titanium plasma-sprayed (TPS; 5.2-microm R(a)). At confluence, cells were treated for 24 h with control media or media containing 10(-7) M 1alpha,25(OH)(2)D(3). One-half of the cultures received 1 microm or 10 microm PD98059, a specific inhibitor of the ERK family of MAPKs. PD98059 alone did not affect proliferation, osteocalcin production, or production of transforming growth factor-beta1 or nitric oxide, regardless of the surface roughness. Alkaline phosphatase was reduced by the inhibition of the ERK family kinases on all surfaces to a comparable extent. However, when PD98059 was added to the cultures with 1alpha,25(OH)(2)D(3), the effects of the seco-steroid were blocked, including the synergistic increases seen in MG63 cells cultured on SLA or TPS. These results indicate that ERK1/2 MAPK is required for the maintenance of alkaline phosphatase at control levels and that the effects of 1alpha,25(OH)(2)D(3) are mediated by ERK1/2. However, the effects of surface roughness are not due to the ERK family of MAPKs. This suggests that alternative pathways may be used, including those mediated by other MAPK subclasses.
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PMID:Osteoblast response to titanium surface roughness and 1alpha,25-(OH)(2)D(3) is mediated through the mitogen-activated protein kinase (MAPK) pathway. 1137 60


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