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 D3, 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

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-β-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

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-β-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

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-β-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

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, PIP5K1 is recruited by the Ecadherin--catenin complex to the plasma membrane where it provides the substrate PIP2 for both PI3K and PLC-1. 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 PIP5K1 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 PIP5K1 activation. PP1 knockdown blocked extracellular calciuminduced increase in serine dephosphorylation and activity of PIP5K1 and induction of keratinocyte differentiation markers. Knockdown of PLC-1, the downstream effector of PIP5K1 , blocked upstream dephosphorylation and PIP5K1 activation induced by calcium. Coimmunoprecipitation revealed calcium induced recruitment of PP1 to the E-cadherin-catenin-PIP5K1 complex in the plasma membrane. These results indicate that PP1 is recruited to the extracellular calcium-dependent E-cadherin-catenin-PIP5K1 complex in the plasma membrane to activate PIP5K1 , which is required for PLC-1 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

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