1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.
1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of lysophospholipid.
Schwartz
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Department of Biomedical Engineering, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA.
Shaked
D
D
Hardin
R R
RR
Gruwell
S
S
Dean
D D
DD
Sylvia
V L
VL
Boyan
B D
BD
D000818:Animals; D019902:Chondrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008246:Lysophospholipids / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D008954:Models, Biological; D010716:Phosphatidylinositols / Q000378:metabolism; D054799:Phospholipase C beta; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology*
C116441:2-methyl-1,25-dihydroxyvitamin D3; D007527:Isoenzymes; D008246:Lysophospholipids; D010716:Phosphatidylinositols; D020033:Protein Isoforms; D014807:Vitamin D; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D054799:Phospholipase C beta; C518035:Plcb1 protein, rat; C518041:Plcb3 protein, rat