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

Abstract

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.

Figure 1

Calcium induces PIP5K1α dephosphorylation. Human keratinocytes cultured with 1.2 mM and harvested at time points indicated and analyzed by western analysis.

Figure 2

Knockdown of PP1 blocks calcium-induced PIP5K1α dephosphorylation. Cultured human keratinocytes were treated with PP1α siRNA, PP1β siRNA, and PP1γ siRNA for 72 hours and then with calcium for 5 min. (a) Cells were harvested and protein levels of PP1α, PP1β, and PP1γ were determined by western analysis. (b) Data expressed are mean ± SD of three separate experiments, ^∗ p < 0.05.

Figure 3

Knockdown of PP1 blocks calcium-induced PIP5K1α activity. Cultured human keratinocytes were treated with siRNA for PP1 for 72 hours and then with calcium for 5 min. Cells were harvested and total cell lysates were isolated for PIP5K1α activity assay. (a) The autoradiograph shown is from a representative experiment repeated thrice with three separate siRNA treatments. (b) The PIP₂ signal intensities were quantitated by Image Pro Plus Software and normalized to band intensities of PIP5K1α in the corresponding western blot. Results are expressed as percentages of the values in the control lane (the presence of 0.03 mM calcium and control siRNA). Data expressed are mean ± SD of three separate experiments, ^∗ p < 0.05 (significantly different from the control in the presence of 0.03 mM calcium and siRNA).

Figure 4

Knockdown of PP1 blocks calcium-induced keratinocyte differentiation. Cultured human keratinocytes were treated with PP1α siRNA, PP1β siRNA, and PP1γ siRNA for 72 hours and then in calcium for 5 min. Cells were harvested and total cell lysates were isolated. (a) The protein levels of PP1α, PP1β, PP1γ, keratin 1, involucrin, and transglutaminase were determined by western analysis. (b) Data expressed are mean ± SD of three separate experiments, ^∗ p < 0.05.

Figure 5

Calcium induces PP1 recruitment to E-cadherin in the plasma membrane. (a) Cultured human keratinocytes were treated with 1.2 mM calcium for 5 min. Cells were harvested and the protein levels of PP1α, PP1β, PP1γ, PIP5K1α, E-cadherin, integrin α2 (plasma membrane marker), BIP (endoplasmic reticulum marker), and GM130 (cis-Golgi marker) in total cell lysate and plasma membrane lysate were determined by western analysis. The results are from a representative experiment that was repeated three times. (b) The total cell lysates and the plasma cell lysates were isolated and analyzed by immunoprecipitation (IP) with antibody against E-cadherin followed by western analysis with antibodies against PP1α, PP1β, PP1γ, PIP5K1α, and E-cadherin.

Figure 6

Inhibition of PI3K, PLC, or PKC activity blocks calcium-induced PIP5K1α dephosphorylation. Cultured human keratinocytes were treated with inhibitors for PI3K, PLC, and PKC for 72 hours and then with calcium for 5 min. Cells were harvested and then analyzed by western analysis.

Figure 7

Inhibition of PI3K, PLC, or PKC activity blocks calcium-induced PIP5K1α activity. Cultured human keratinocytes were treated with inhibitors for PI3K, PLC, and PKC for 72 hours and then with calcium for 5 min. Cells were harvested and total cell lysates were isolated for PIP5K1α activity assay. (a) The autoradiograph shown is from a representative experiment repeated thrice with three separate siRNA treatments. (b) The PIP₂ signal intensities were quantitated by Image Pro Plus Software and normalized to band intensities of PIP5K1α in the corresponding western blot. Results are expressed as percentages of the values in the control lane (the presence of 0.03 mM calcium and control siRNA). Data expressed as mean ± SD of three separate experiments, ^∗ p < 0.05 (significantly different from the control in the presence of 0.03 mM calcium and siRNA).

Figure 8

Knockdown of E-cadherin/catenins or PLC-γ1 blocks calcium-induced PIP5K1α dephosphorylation. Cultured human keratinocytes were treated with E-cadherin siRNA, β-catenin siRNA, p120 siRNA, and PLC-γ1 siRNA for 72 hours and then in calcium for 5 min. Cells were harvested and total cell lysates were isolated. The protein levels of E-cadherin, β-catenin, p120, PLC-γ1, p-PIP5KIα (serine), and PIP5KIα were analyzed by western analysis. Data expressed are mean ± SD of three separate experiments, ^∗ p < 0.05.

Figure 9

Knockdown of E-cadherin/catenins or PLC-γ1 blocks calcium-induced PIP5K1α activity. Cultured human keratinocytes were treated with siRNA for E-cadherin, β-catenin, p120, and PLC-γ1 for 72 hours and then with calcium for 5 min. Cells were harvested and total cell lysates were isolated for PIP5K1α activity assay. (a) The autoradiograph shown is from a representative experiment repeated thrice with three separate siRNA treatments. (b) The PIP₂ signal intensities were quantitated by Image Pro Plus Software and normalized to band intensities of PIP5K1α in the corresponding western blot. Results are expressed as percentages of the values in the control lane (the presence of 0.03 mM calcium and control siRNA). Data are expressed as mean ± SD of three separate experiments, ^∗ p < 0.05 (significantly different from the control in the presence of 0.03 mM calcium and siRNA).

Figure 10

The proposed model. A proposed model for the signaling pathway of calcium-induced keratinocyte differentiation. In high calcium conditions, PP1 is recruited to the calcium-dependent E-cadherin-β-catenin-PIP5K1α complex in the plasma membrane to activate PIP5K1α, which increases the synthesis of the substrate PIP₂ for both PLC-γ1 and PI3K. PLC-γ1 hydrolyzes PIP₂ to IP₃, which in turn increases calcium concentration leading to keratinocyte differentiation.