p38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

Abstract

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

“…Cip1 promoter, leading to increased p21 Cip1 expression and reduced cell proliferation (2,34). However, less is known about mechanisms that antagonize this action to maintain keratinocyte proliferative potential.…”

“…PIHP01250) in KSFM. After 24 h, the cells were shifted to Epilife medium containing 1.4 mM calcium chloride and 5 g/ml vitamin C and cultured at the air-liquid interface (3,34). Fresh Epilife medium was added every 2 days.…”

“…For protein lysates, the inserts were washed twice with phosphate-buffered saline, and the cells were harvested in 0.0625 M Tris-HCl (pH 7.5) containing 10% glycerol, 5% SDS, and 5% ␤-mercaptoethanol. Then the cells were sonicated and centrifuged at 10,000 rpm for 5 min, and the supernatant was collected for immunoblot analysis (34).…”

Multifunctional roles of PKCδ: Opportunities for targeted therapy in human disease

“…Other studies support a role for PKC isoforms in cell cycle arrest (Watanabe et al, 1992) and specifically PKCδ in mediating G1 arrest through induction of p21 (Nakagawa et al, 2005), in S phase arrest (Santiago-Walker et al, 2005), and in the maintenance of the G2/M DNA damage checkpoint (LaGory et al, 2010). Finally, PKCδ may regulate the transcription of apoptotic genes and p21 through activation of p53 (Nakagawa et al, 2005; Perletti et al, 2005; Ryer et al, 2005; Yoshida, Liu, & Miki, 2006; Liu, Lu, Miki, & Yoshida, 2007; Saha, Adhikary, Kanade, Rorke, & Eckert, 2014). …”

MEKK1-dependent phosphorylation of calponin-3 tunes cell contractility

“…We postulated that MEKK1 might be involved in the positivefeedback mechanism that mediates substrate-rigidity-dependent regulation of cell contractility (Giannone and Sheetz, 2006;Buxboim et al, 2010;Trichet et al, 2012), and examined whether the activity of MEKK1 was modulated by actomyosin contraction, the major source of cellular contractility (Beningo et al, 2006). MEKK1 activity was assessed by evaluating the level of MEKK1 phosphorylation at Thr1381 in its activation loop, the autophosphorylation that reportedly represents MEKK1 activation (Matsuzawa et al, 2008;Enzler et al, 2009;Saha et al, 2014). In an immunoblot analysis of C2C12 cell lysate using an antibody that was raised against Thr1381-phosphorylated MEKK1 (Fig.…”