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.
Protein arginine methyltransferase 5 (PRMT5) cooperates with methylosome protein 50 (MEP50) to arginine methylate histone H3 and H4 to silence gene expression, and increased PRMT5 activity is associated with enhanced cancer cell survival. We have studied the role of PRMT5 and MEP50 in epidermal squamous cell carcinoma. We show that knockdown of PRMT5 or MEP50 results in reduced H4R3me2s formation, and reduced cell proliferation, invasion, migration and tumor formation. We further show that treatment with sulforaphane (SFN), a cancer preventive agent derived from cruciferous vegetables, reduces PRMT5 and MEP50 level and H4R3me2s formation, and this is associated with reduced cell proliferation, invasion and migration. The SFN-dependent reduction in PRMT5 and MEP50 level requires proteasome activity. Moreover, SFN-mediated responses are partially reversed by forced PRMT5 or MEP50 expression. SFN treatment of tumors results in reduced MEP50 level and H4R3me2s formation, confirming that that SFN impacts this complex in vivo. These studies suggest that the PRMT5/MEP50 is required for tumor growth and that reduced expression of this complex is a part of the mechanism of SFN suppression of tumor formation.
Abstract. Epigenetics is an important emerging area for study of mechanisms of cancer prevention. In recent years, it has been realized that cancer prevention agents, derived from natural dietary sources, impact cancer cell survival by modulating epigenetic processes. In the present manuscript, we review key epigenetic regulatory mechanisms and examine the impact of sulforaphane and green tea polyphenols on these processes. We also discuss available information on the epigenetics in the context of skin cancer. These studies indicate that diet-derived chemopreventive agents modulate DNA methylation status and histone modification via multiple processes and point to additional areas for study of epigenetic mechanisms in skin cancer.
PKCδ and p38δ are key proteins in a cascade that stimulates keratinocyte differentiation. This cascade activates transcription of involucrin (hINV) and other genes associated with differentiation. Protein arginine methyltransferase 5 (PRMT5) is an arginine methyltransferase that symmetrically dimethylates arginine residues. This protein interacts with a cofactor, MEP50, and symmetrically dimethylates arginine eight of histone 3 (H3R8me2s) and arginine three of histone 4 (H4R3me2s) to silence gene expression. We use the involucrin gene as a tool to understand the relationship between PKCδ/p38δ and PRMT5/MEP50 signaling. MEP50 suppresses hINV mRNA level and promoter activity. This is associated with increased arginine dimethylation of hINV gene-associated H3/H4. We further show that the PKCδ/p38δ keratinocyte differentiation cascade reduces PRMT5 and MEP50 expression, association with the hINV gene promoter, and H3R8me2s and H4R2me2s formation. We propose that PRMT5/MEP50-dependent methylation is an epigenetic mechanism that assists in silencing of hINV expression, and that PKCδ signaling activates gene expression by directly activating transcription and by suppressing PRMT5/MEP50 dependent arginine dimethylation of promoter associated histones. This is an example of crosstalk between PKCδ/p38δ signaling and PRMT5/MEP50 epigenetic silencing.
Background: Keratinocytes cease proliferation during differentiation, and the mechanism that mediates these events is not well understood.
Results: PRMT5/MEP50 control p21Cip1 promoter methylation to silence gene expression, and this is reversed by PKC␦/p38␦ signaling. Conclusion: PRMT5/MEP50 and PKC␦/p38␦ signaling produce opposing actions to control keratinocyte proliferation during differentiation. Significance: This study describes cross-talk between MAPK signaling and epigenetic mechanisms to control cell proliferation.
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