Phospholipase C β and Membrane Action of Calcitriol and Estradiol

Abstract

We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca 2؉ ] 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 [Ca 2؉ ] i increase, we described different PLC isoforms (␤1, ␤2, ␥1, and ␥2, but not ␤4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C ␤ was involved in the mobilization of Ca 2؉ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17␤ estradiol, and PLC ␥ was ineffective. The data also showed that only a PLC ␤1 linked to a Pertussis toxin-insensitive G-protein and a PLC ␤2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17␤ estradiol on the mobilization of Ca 2؉ from intracellular Ca 2؉ 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.An increase in the turnover of inositol lipids in response to receptor is one of the most important molecular mechanisms used by cells for transmembrane signaling. The initial event is the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a reaction catalyzed by a phosphoinositide-specific phospholipase C (PLC), 1 which generates two intracellular second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol (1-4). Inositol 1,4,5-trisphosphate binds to specific receptors on the endoplasmic reticulum (5) and mobilizes intracellular calcium, whereas diacylglycerol activates protein kinase C (6), which results in increased phosphorylation of cellular proteins.Molecular cloning has revealed at least three major families of PLC, ␤, ␥, and ␦ (7-9). Each of these families occurs in a number of isoforms. The enzymes are classified on the basis of their size and their immunological and structural similarities. The PLC isoforms have two highly conserved domains, X and Y, which form the active site of the protein. PLC ␦ and PLC ␥ proteins differ from PLC ␤ in that they have shorter C-terminal extensions past the end of the Y domain (9). This diversity among the PLC isoforms also extends to distinct mechanisms of regulation and function for the three PLC families. PLC ␥ is regulated via the phosphorylation of tyrosine residues between the X and Y domains by receptor tyrosine kinases (10 -12). PLC ␤ enzymes, of which there are four isoforms, PLC ␤1-4, are regulated via heterotrimeric G-proteins in response to an agonist binding to a receptor (13-15). The way in which PLC ␥ is regulated is not yet known, but enzyme activity is not affected by either the G-protein subunits or by receptor tyrosine kinases (16).The activat...

Acute effects of 17β-estradiol on femoral veins from adult gonadally intact and ovariectomized female pigs

“…It could not be determined from this study whether 17␤-estradiol binds to a putative plasma membrane receptor (33) and/or other proteins to cause acute, nongenomic relaxations to 17␤-estradiol in femoral veins. Nongenomic actions of estrogen in other tissues include increases in cAMP, mitogen-activated protein kinase activity, phospholipase C (28,39,51), and activation of Maxi-K (hSlo) and L-type Ca 2ϩ channels (26,53).…”

Steroid hormone regulation of Clca3 expression in the murine uterus

“…Even though the 319 bp promoter fragment did not contain a full ERE consensus site, E2 induction was still observed, albeit significantly reduced. This residual regulation by E2 may be through activation of the ERE half-site at -176 -171, or it may be through a mechanism independent of DNA binding (Aronica et al 1994, Tesarik & Mendoza 1995, Le Mellay et al 1997, Simoncini et al 2004. Interestingly, the 0·5 kb region displays higher luciferase activity than the 1·0 kb full-length promoter region.…”

The Rapid Effects of 1,25-Dihydroxyvitamin D3 Require the Vitamin D Receptor and Influence 24-Hydroxylase Activity

“…The observed clear differences in the calcium responses to 1,25-(OH) 2 D 3 of the two mutated cell types offer the most valuable tools to search for the long term consequences of these rapid effects. At present, several rapid effects of 1,25-(OH) 2 D 3 have been reported on intracellular calcium entry and distribution, on the opening of Ca 2ϩ and Cl Ϫ channels, and on protein kinase C, MAPK, phospholipase A2, phospholipase C-␤1, and G␣q/11 activation (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). These rapid effects are thought to be involved in the process of cell proliferation/differentiation but also in vitamin D metabolism and catabolism via a MAPK-mediated stimulation of 25-(OH)D 3 -24-hydroxylase gene expression (7,31).…”

“…Calcium Measurement-Confluent fibroblasts were incubated for 24 h in a serum-free medium and assayed for intracellular calcium concentration ([Ca 2ϩ ] i ) as described (8). In brief, cells were loaded with 1 M Fura-2/AM in Hanks' HEPES, pH 7.4 (137 mM NaCl, 5.6 mM KCl, 0.441 mM KH 2 PO 4 , 0.442 mM Na 2 HPO 4 , 0.885 mM MgSO 4 .7H 2 O, 27.7 mM glucose, 1.25 mM CaCl 2 , and 25 mM HEPES) for 30 min at room temperature.…”

“…Although most classical effects of 1,25-(OH) 2 D 3 involve this genomic pathway, several other effects may be observed very rapidly, within seconds to minutes, and are not blocked by inhibitors of transcription or translation, suggesting a more direct action of the hormone at the membrane level (3)(4)(5)(6)(7). These effects include activation of the phospholipase C pathway via G␣ q/11 (8,9), activation of the adenylate cyclase pathway (10), opening of voltage-gated Ca 2ϩ channels (L-type) in the plasma membrane (11,12), Ca 2ϩ mobilization from the endoplasmic reticulum (5), and an increase in cyclic guanosine monophosphate levels (13). The rapid effects of 1,25-(OH) 2 D 3 also include the activation of the mitogenactivated protein kinase (MAPK) pathway (14 -16), which induces a cross-talk with the cell nucleus via phosphorylation of cytoplasmic kinases and nuclear transcription factors (7,16,17).…”