Critical role for the catalytic activity of phospholipase C-gamma1 in epidermal growth factor-induced cell migration.

Phospholipase C-gamma1 (PLC-gamma1), a tyrosine kinase substrate, has been implicated in the pathway for the epidermal growth factor receptor (EGFR)-induced cell migration. However, the underlying mechanism by which PLC-gamma1 mediates EGFR-induced cell migration remains elusive. In the present study, we sought to determine whether the lipase activity of PLC-gamma1 is required for EGFR-induced cell migration. We found that overexpression of PLC-gamma1 in squamous cell carcinoma SCC4 cells markedly enhanced EGF-induced PLC-gamma1 activation, intracellular calcium rise, and cell migration. This enhancement was abolished by mutational inactivation of the catalytic domain of PLC-gamma1. Inhibition of the downstream signaling processes mediated by the activity of phospholipase C (PLC) using IP(3) receptor inhibitor or intracellular calcium chelator blocked EGF-induced cell migration. These data indicate that EGF-induced cell migration is mediated by the lipase domain of PLC-gamma1 and the subsequent IP(3) generation and intracellular calcium mobilization.

The SH3 domain, but not the catalytic domain, is required for phospholipase C-gamma1 to mediate epidermal growth factor-induced mitogenesis.

Phospholipase C-gamma1 (PLC-gamma1) is a multiple-domain protein and plays an important role in epidermal growth factor (EGF)-induced cell mitogenesis, but the underlying mechanism is unclear. We have previously demonstrated that PLC-gamma1 is required for EGF-induced mitogenesis of squamous cell carcinoma (SCC) cells, but the mitogenic function of PLC-gamma1 is independent of its lipase activity. Earlier studies suggest that the Src homology 3 (SH3) domain of PLC-gamma1 possesses mitogenic activity. In the present study, we sought to determine the role of the SH3 domain of PLC-gamma1 in EGF-induced SCC cell mitogenesis. We examined the effect of overexpression of PLC-gamma1, a catalytically active PLC-gamma1 mutant lacking the SH3 domain or a catalytically inactive PLC-gamma1 mutant lacking the X domain on EGF-induced SCC4 (tongue squamous cell carcinoma) cell mitogenesis. We found that overexpression of PLC-gamma1 enhanced EGF-induced SCC4 cell mitogenesis. This enhancement was abolished by deletion of the SH3 domain but not by deletion of the X catalytic domain. These data suggest that the SH3 domain, but not the catalytic domain, is required for PLC-gamma1 to mediate EGF-induced SCC4 cell mitogenesis.

Phospholipase C-gamma1 is required for the epidermal growth factor receptor-induced squamous cell carcinoma cell mitogenesis.

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-gamma1 (PLC-gamma1) is a downstream target of EGFR signaling, but the role and necessity of PLC-gamma1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-gamma1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-gamma1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-gamma1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-gamma1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-gamma1 is independent of its lipase activity.

Phosphoprotein Phosphatase 1 Is Required for Extracellular Calcium-Induced Keratinocyte Differentiation.

Extracellular calcium is a major regulator of keratinocyte differentiation in vitro and appears to play that role in vivo, but the mechanism is unclear. We have previously demonstrated that, following calcium stimulation, PIP5K1α is recruited by the E-cadherin-ß-catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-γ1. This signaling pathway is critical for calcium-induced generation of second messengers including IP3 and intracellular calcium and keratinocyte differentiation. In this study, we explored the upstream regulatory mechanism by which calcium activates PIP5K1α and the role of this activation in calcium-induced keratinocyte differentiation. We found that treatment of human keratinocytes in culture with calcium resulted in an increase in serine dephosphorylation and PIP5K1α activation. PP1 knockdown blocked extracellular calcium-induced increase in serine dephosphorylation and activity of PIP5K1α and induction of keratinocyte differentiation markers. Knockdown of PLC-γ1, the downstream effector of PIP5K1α, blocked upstream dephosphorylation and PIP5K1α activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1α complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which is required for PLC-γ1 activation leading to keratinocyte differentiation.

Lowered humidity produces human epidermal equivalents with enhanced barrier properties.

Multilayered human keratinocyte cultures increasingly are used to model human epidermis. Until now, studies utilizing human epidermal equivalents (HEEs) have been limited because previous preparations do not establish a normal epidermal permeability barrier. In this report, we show that reducing environmental humidity to 50% relative humidity yields HEEs that closely match human postnatal epidermis and have enhanced repair of the permeability barrier. These cultures display low transepidermal water loss and possess a calcium and pH gradient that resembles those seen in human epidermis. These cultures upregulate glucosylceramide synthase and make normal-appearing lipid lamellar bilayers. The epidermal permeability barrier of these cultures can be perturbed, using the identical tools previously described for human skin, and recover in the same time course seen during in vivo barrier recovery. These cultures will be useful for basic and applied studies on epidermal barrier function.

Phosphatidylinositol-4-phosphate 5-kinase 1alpha mediates extracellular calcium-induced keratinocyte differentiation.

Extracellular calcium (Cao) is a major regulator of keratinocyte differentiation, but the mechanism is unclear. Phosphatidylinositol-4-phosphate 5-kinase 1alpha (PIP5K1alpha) is critical in synthesizing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. In this study, we sought to determine whether PIP5K1alpha plays a role in mediating the ability of Cao to induce keratinocyte differentiation. We found that treatment of human keratinocytes in culture with Cao resulted in increased PIP5K1alpha level and activity, as well as PI(4,5)P2 level, binding of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] to and activation of phospholipase C-gamma1 (PLC-gamma1), with the resultant increase in inositol 1,4,5-trisphosphate (IP3) and intracellular calcium (Cai). Knockdown of PIP5K1alpha in human keratinocytes blocked Cao-induced increases in the binding of PI(3,4,5)P3 to PLC-gamma1; PLC-gamma1 activity; levels of PI(4,5)P2, IP3, and Cai; and induction of keratinocyte differentiation markers. Coimmunoprecipitation and confocal studies revealed that Cao stimulated PIP5K1alpha recruitment to the E-cadherin-catenin complex in the plasma membrane. Knockdown of E-cadherin or beta-catenin blocked Cao-induced activation of PIP5K1alpha. These results indicate that after Cao stimulation PIP5K1alpha is recruited by the E-cadherin-catenin complex to the plasma membrane where it provides the substrate PI(4,5)P2 for both PI3K and PLC-gamma1. This signaling pathway is critical for Cao-induced generation of the second messengers IP3 and Cai and keratinocyte differentiation.

Regulation of human epidermal keratinocyte differentiation by the vitamin D receptor and its coactivators DRIP205, SRC2, and SRC3.

It has long been known that the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3), stimulates differentiation and inhibits proliferation in epidermal keratinocytes through interaction with the vitamin D receptor (VDR). VDR functions through the coordinate binding of vitamin D response elements in the DNA and specific coactivator proteins which help to initiate transcription. It was recently observed that VDR binds to two major coactivator complexes, DRIP (VDR-interacting protein) and SRC (steroid receptor coactivator), during keratinocyte differentiation. To determine the role of VDR and its coactivators in mediating keratinocyte differentiation, we developed an adenoviral system to knock down, or in the case of VDR, overexpress these genes. In order to study all stages of keratinocyte development, we employed an advanced differentiated normal human keratinocyte culture system that produces a multilayer phenotype similar to that of normal skin. These studies have shown that VDR, DRIP, and SRC are all required for promotion of both early and late keratinocyte differentiation. Additionally, each individual differentiation marker that was assayed has a different specificity for the coactivators that regulate its expression.