The calcium/calcineurin pathway promotes hemidesmosome stability through inhibition of β4 integrin phosphorylation

Abstract

Cell migration depends on cells being able to create and disassemble adhesive contacts. Hemidesmosomes are multiprotein structures that attach epithelia to basal lamina and disassemble during migration and carcinoma invasion. Phosphorylation of the β4 integrin, a hemidesmosome component, induces disassembly. Although kinases involved in β4 phosphorylation have been identified, little is known about phosphatases countering kinase action. Here we report that calcineurin, a serine-threonine protein phosphatase, regulates β4 phosphorylation. Calcineurin inhibitor cyclosporin A (CsA) and calcineurin-siRNA increase β4 phosphorylation, induce hemidesmosome disassembly, and increase migration in HaCat keratinocytes, suggesting that calcineurin negatively regulates β4 phosphorylation. We found no direct dephosphorylation of β4 by calcineurin or association between β4 and calcineurin, suggesting indirect regulation of β4 phosphorylation. We therefore assessed calcineurin influence on MAPK and PKC, known to phosphorylate β4. CsA increased MAPK activity, whereas MAPK inhibitors reduced CsA-induced β4 phosphorylation, suggesting that calcineurin restricts β4 phosphorylation by MAPK. Calcineurin is activated by calcium. Increased [Ca(2+)](i) reduces β4 phosphorylation and stabilizes hemidesmosomes, effects that are reversed by CsA, indicating that calcineurin mediates calcium effects on β4. However, MAPK activation is increased when [Ca(2+)](i) is increased, suggesting that calcineurin activates an additional mechanism that counteracts MAPK-induced β4 phosphorylation. Interestingly, in some squamous cell carcinoma cells, which have reduced hemidesmosomes and increased β4 phosphorylation, an increase in [Ca(2+)](i) using thapsigargin, bradykinin, or acetylcholine can increase hemidesmosomes and reduce β4 phosphorylation in a calcineurin-dependent manner. These findings have implications in calcineurin-inhibitor induced carcinoma, a complication of immunosuppressive therapy.

FIGURE 1.

CN regulates β4 phosphorylation and HD stability. A, effects of CN inhibitor CsA on β4 phosphorylation (Ser¹³⁵⁶). HaCat cells were treated for different lengths of time with CsA (1 μm), EGF (50 ng/ml), or a combination of both and were analyzed by Western blot. B, effects of CsA on HD stability. Quantitative IF analysis of detergent-resistant β4 was used to estimate HD-LS. Cells were treated with CsA (1 μm), EGF at low concentration (1 ng/ml), or a combination of both for the indicated time intervals and then detergent-extracted, fixed, and processed for IF using β4 Ab to measure fluorescence-integrated density per cell. C, representative IF images quantified in B. Bar = 10 μm. D, effects of CsA on EGF-induced migration. Cell migration was tested using wound healing assays in the presence or absence of EGF (1 ng/ml) and/or CsA (*, p < 0.05 versus control; **, p < 0.05 versus EGF or CsA). E, effects of CN-siRNA on β4 phosphorylation. HaCat keratinocytes were transfected with CN- or negative control siRNA and analyzed after 48 h for β4 phosphorylation by Western blot. F, effects of CN-siRNA on HD-LS. Control or CN-siRNA transfectants were analyzed for HD-LS abundance using quantitative IF (*, p < 0.05 versus control siRNA; **, p < 0.05 versus control siRNA+EGF or CN-siRNA). G, effects of CN-siRNA on keratinocyte migration using wound healing assays. HaCat keratinocytes transfected with control or CN-siRNA were grown to confluency for 48 h before performing wound healing assays in the presence of EGF (1 ng/ml) for 18 h. (*, p < 0.05 versus control siRNA; **, p < 0.05 versus control siRNA+EGF or CN-siRNA)

FIGURE 2.

CN regulates β4 phosphorylation through MAPK- and PKC-dependent pathways. A, HaCat keratinocytes were preincubated or not in the presence of MAPK inhibitor U0126 (10 μm) or PKC inhibitor Go6983 (1 μm) for 20 min before adding CsA for an additional 20 min and analyzed by Western blot using the indicated Abs. pS₁₃₅₆, Ser(P)¹³⁵⁶; pERK, phospho-ERK. B, time course analysis of MAPK pathway activation. HaCat keratinocytes were treated with CsA (1 μm), EGF (50 ng/ml), or a combination and then stained with phospho-ERK1/2 or total ERK Abs and analyzed by Western blot.

FIGURE 3.

Ca²⁺ signaling regulates β4 phosphorylation and HD stability through CN. A, effects of [Ca²⁺]_i modulation on EGF-induced phosphorylation of β4. HaCat keratinocytes were preincubated in the presence or not of THG (1 μm), which increases [Ca²⁺]_i, or BAPTA-AM (10 μm), which reduces [Ca²⁺]_i, and then stimulated or not with EGF for 20 min. Cell lysates were analyzed with the indicated Abs. pS₁₃₅₆, Ser(P)¹³⁵⁶.B, inhibition of EGF-induced β4 phosphorylation by THG is reversed by CsA. Cells were preincubated with or without THG and/or CsA for 10 min before stimulation with EGF (50 ng/ml) for 20 min. pERK, phospho-ERK. C, effects of [Ca²⁺]_i modulation on HD stability. HaCat keratinocytes were preincubated in the presence or not of THG, CsA, or BAPTA-AM, before stimulation with EGF for 20 min, then processed for quantitative IF analysis of HD-LS (*, p < 0.05 versus control; **, p < 0.05 between bracketed columns). D, effects of [Ca²⁺]_i modulation on β4 phosphorylation induced by PKC. Cells were preincubated in the presence or not of THG for 10 min before stimulation with phorbol 12-myristate 13-acetate (PMA) (25 ng/ml) for 30 min. E, MAPK dependence of BAPTA effects on β4 phosphorylation. Cells were preincubated with MAPK inhibitor U0126 for 20 min and then treated with BAPTA-AM and analyzed by Western blot. F, analysis of calmodulin inhibition on β4 phosphorylation. Cells were preincubated in the presence or not of calmodulin inhibitor W13 (15 μg/ml) and/or THG for 20 min before stimulation with EGF for 20 min and analyzed by Western blot.

FIGURE 4.

Increased [Ca²⁺]_i in SCC cells stabilizes HD and reduces β4 phosphorylation and migration potential. A, SCC cells were incubated in the presence or absence of THG and/or CsA for 20 min as indicated and analyzed by Western blot. pS₁₃₅₆, Ser(P)¹³⁵⁶. B and C, IF analysis of HD-LS (B) or wound healing assay (C) of SCC cells incubated in the presence or absence of THG. D, CN activity analysis of SCC cells. Cell lysates obtained from SCC cell lines were analyzed for CN activity using a colorimetric assay kit. E, THG effects on β4 phosphorylation and ERK1/2 activation in A431 cells. Cells were incubated in the presence or absence of THG for 20 min and analyzed by Western blot (*, p < 0.05 versus respective controls). pERK, phospho-ERK.

FIGURE 5.

BK induces CN-dependent inhibition of β4 phosphorylation and stabilizes HD in keratinocytes and SCC; effects on migration are cell type-dependent. A, BK modulation of EGF effects on β4 phosphorylation. HaCat cells were preincubated in the presence or not of BK (0.1 μm) before stimulating with EGF(50 ng/ml) for the indicated times. pS₁₃₅₆, Ser(P)¹³⁵⁶; pERK, phospho-ERK. B, BK modulation of EGF effects on HD-LS stability. HaCat cells were preincubated in the presence or not of BK before stimulating with EGF for 10 min and being processed for HD-LS analysis (*/**, p < 0.05 between bracketed columns). C, effects of BK on β4 phosphorylation in SCC cells. SCC cells were treated for the indicated times with BK and processed for Western analysis using the indicated Abs. D, dependence of BK effects on CN. SCC cells were preincubated in the presence or not of CsA for 10 min and then stimulated with BK for 5 min. E, effects of BK on HD-LS stability versus b4 phosphorylation in SCC cells. SCC cells were treated with BK for the indicated times and then processed for a HD-LS analysis using quantitative IF (relative fluorescence-integrated density) or processed for Western analysis (relative densitometric ratios). pS, phospho-serine. F, effects of BK on cell migration. Wound healing assays were performed on HaCat keratinocytes or SCC Colo-16 cells in the presence or absence of BK, and closure of the wound was measured as described under “Experimental Procedures” (*, p < 0.05 versus respective controls).

FIGURE 6.

caCN-Myc inhibits β4 phosphorylation and increases HD stability. A, Cos7 cells cotransfected with α6, β4, + or − caCN-Myc, or mock plasmid (Mock) were treated with EGF and analyzed by Western blot. pS₁₃₅₆, Ser(P)¹³⁵⁶. B, HaCat keratinocytes or SCC cells were transfected with caCN-Myc and processed for IF analysis using the indicated Abs (green, anti-β4; red, anti-Ser(P)¹³⁵⁶; blue, anti-Myc). HaCat cells were incubated in serum-containing medium to induce phosphorylation of the β4 integrin, whereas SCC cells were in serum-free medium. Notice that Myc-positive cells (cyan outline) have reduced phosphorylation of the β4 integrin, whereas preserving or increasing HD-LS characteristic plaques (yellow arrowheads) when compared with the Myc-negative neighbor cells (magenta arrowheads). Bar = 10 μm.