Phospholipase C-γ1 Is Required for Calcium-induced Keratinocyte Differentiation

Abstract

Phospholipase C-␥1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by calcium. Phospholipase C-␥1, therefore, may be involved in the signal transduction system leading to calcium regulation of keratinocyte differentiation. To test this hypothesis, expression of phospholipase C-␥1 in human keratinocytes was blocked by transfecting cells with the antisense human phospholipase C-␥1 cDNA construct. These cells demonstrated a dramatic reduction in phospholipase C-␥1 protein level compared with the empty vector-transfected cells and a marked reduction in the mRNA and protein levels of the differentiation markers involucrin and transglutaminase following administration of calcium. Similarly, cotransfection of antisense phospholipase C-␥1 constructs with a luciferase reporter vector containing involucrin or transglutaminase promoters led to a substantial reduction in calcium-stimulated involucrin and transglutaminase promoter activities. Similar results were seen following treatment with a specific phospholipase C inhibitor U73122. To determine whether phospholipase C-␥1 regulated differentiation by controlling intracellular calcium, we examined the ability of antisense phospholipase C-␥1 to block the calcium-induced rise in intracellular calcium and found that it could. These findings indicate that phospholipase C-␥1 is a critical component of the signaling pathway mediating calcium regulation of keratinocyte differentiation via its mobilization of intracellular calcium.A variety of extracellular stimuli exert their physiologic effects by activating phospholipase C (PLC) 1 isozymes. PLC activation results in hydrolysis of phosphatidylinositol bisphosphate (PIP 2 ), a cell membrane phospholipid, generating inositol trisphosphate (IP 3 ), which releases calcium from intracellular stores, and diacylglycerol (DG), which activates protein kinase C. These second messengers subsequently activate a number of cellular reactions leading to changes in cell proliferation and differentiation (1, 2). There are at least three known types of PLC (-␤, -␥, and -␦), and each type has a number of subtypes (3, 4). The ␥-type enzymes are unique in that they contain SH 2 and SH 3 domains and are subject to activation by a number of growth factor receptors such as receptors for epidermal growth factor, nerve growth factor, and plateletderived growth factor (5-8).Human keratinocytes possess a functionally active inositol lipid signaling system (9 -12) that may play an essential role in keratinocyte differentiation (13-15). The addition of calcium leads to keratinocyte differentiation and the activation of several signaling pathways, one of which involves PLC-␥1, the predominant isoform of PLC in keratinocytes (13,14,16,17). However, it is not clear whether PLC-␥1 plays an essential role in the regulation of keratinocyte differentiation by calcium. To address this issue, we blocked PLC-␥1 production with antisense PLC-␥1 cDNA and inhibited its activity with a specific PLC inhibitor. Our results indicate...

The Recruitment of Phosphatidylinositol 3-Kinase to the E-cadherin-Catenin Complex at the Plasma Membrane Is Required for Calcium-induced Phospholipase C-γ1 Activation and Human Keratinocyte Differentiation

“…Taken together with our previous findings (15,16,55), calcium induced E-cadherin-␤-catenin-p120-catenin complex recruits PI3K via ␤-catenin to the plasma membrane and acti-vates the kinase. PLC-␥1 activated by PIP 3 produced by PI3K in the plasma membrane triggers keratinocyte differentiation through increasing intracellular calcium concentration.…”

“…Indeed, translocation to the membrane alone is sufficient to activate PI3K (25). The activated PI3K converts PIP 2 to PIP 3 , which then activates PLC-␥1 at the plasma membrane to trigger keratinocyte differentiation via increasing intracellular calcium (15,16). Our data FIGURE 4.…”

“…Diacylglycerol remains in the inner layer of the plasma membrane. IP 3 diffuses through the cytosol and binds to the IP 3 receptors in the endoplasmic reticulum and Golgi causing the intracellular calcium rise, which triggers keratinocyte differentiation (15,16,55). In cultured human or mouse keratinocytes activities of PI3K and PLC-␥1 are induced by calcium within minutes and further stimulated up to 1 h before the onset of keratinocyte differentiation.…”

“…This pathway is mediated by PI3K-dependent activation of PLC-␥1, which induces an intracellular calcium rise (15,16). Extracellular calcium activates PI3K, which phosphorylates PIP 2 to generate PIP 3 at the plasma membrane.…”

“…Each type contains several subtypes (14). We have previously demonstrated that PLC-␥1 is required for the extracellular calcium-induced intracellular calcium rise and differentiation of human keratinocytes (15). PLC-␥1 activation and human keratinocyte differentiation induced by calcium require phosphatidylinositol 3-kinase (PI3K) activation (16).…”