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

Abstract

Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21(Cip1) expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21(Cip1) expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21(Cip1) promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21(Cip1) gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21(Cip1) promoter, and that this is associated with enhanced p21(Cip1) expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation.

Keywords: cancer; cell cycle; cyclin; p38 MAPK; protein arginine N-methyltransferase 5 (PRMT5); protein kinase C (PKC).

FIGURE 1.

MEP50 is expressed in the human epidermis and in cultured keratinocytes. A, foreskin tissue sections were stained with anti-MEP50, and binding was visualized with peroxidase-conjugated secondary antibody (top panels). The control is IgG. The arrows indicate MEP50 nuclear localization in suprabasal keratinocytes. Foreskin tissue sections (bottom panels) were fixed and stained with anti-MEP50, and antibody binding was visualized using a FITC-conjugated secondary antibody. The arrows indicate nuclear MEP50 accumulation. Scale bars = 10 μm. B, colocalization of endogenous and expressed MEP50. KERn were electroporated with 3 μg of pcDNA3 or pcDNA3-FLAG-MEP50. After 48 h, protein lysates were tested by immunoblot using anti-FLAG and anti-MEP50. β-actin was used as the loading control. After 48 h, the cells were fixed and costained with anti-FLAG (green) and anti-MEP50 (red). Similar results were observed in each of three experiments. The staining indicates MEP50 distribution in the nucleus and cytoplasm. Scale bars = 10 μm.

FIGURE 2.

MEP50 and PRMT5 are required for keratinocyte proliferation. A, KERn were electroporated twice with control, MEP50, or PRMT5 siRNA, and 15,000 cells/well were plated. After overnight attachment, the cell number was determined (day 0) and at the indicated times thereafter. The values are mean ± S.E. (n = 3). *, p < 0.005. B, immunoblot detection of MEP50 and PRMT5. The immunoblot confirms a reduction in MEP50 in MEP50 siRNA-treated cultures. PRMT5 is also reduced.

FIGURE 3.

MEP50 regulation of p21^Cip1 promoter activity: a role for p53. A, MEP50 suppresses p21^Cip1 expression. KERn were electroporated with the indicated plasmid, and, after 24 h, extracts were prepared for quantitative RT-PCR detection of p21^Cip1 mRNA and immunoblot detection of p21^Cip1 and MEP50. EV, empty vector. B, opposing action of MEP50 and p53. A schematic of the human p21^Cip1 promoter shows the Sp1 and p53 transcription factor response elements. The numbers indicate the distance in nucleotides relative to the transcription start site. KERn were transfected with 0.5 μg of p21-2316, which encodes the full-length wild-type p21^Cip1 promoter, or the promoter harboring mutations at the p53-1 or p53-2 sites, linked to luciferase. After 24 h, extracts were prepared for the luciferase activity assay. The values are mean ± S.E. (n = 3). C–E, KERn were transfected with 0.5 μg of PG13-Luc (the p53 gene promoter linked to luciferase) or p21-2326 and the indicated micrograms of pcDNA3 or pcDNA3-FLAG-MEP50. 48 h post-transfection, cell extracts were prepared and assayed for promoter activity. The values are mean ± S.E. (n = 3). *, p < 0.005.

FIGURE 4.

MEP50 knockdown reduces MEP50 and PRMT5 association at the p21^Cip1 promoter. A and B, KERn were electroporated with 3 μg of control siRNA or MEP50 siRNA. After 48 h, extracts were prepared for ChIP analysis. DNA from 1 million cells was sheared, and 50,000 cell equivalents of DNA were used for immunoprecipitation. The primers span the p21^Cip1 promoter region that includes the p53-2 site. The values are mean ± S.E. (n = 3). *, p < 0.005. C, extracts were prepared from the electroporated cells after 48 h to confirm MEP50 knockdown.

FIGURE 5.

PKCδ and p38δ regulate MEP50 and PRMT5 level and activity. A, KERn were infected with tAd5-EV, Ad5-PKCδ, or tAd5-HA-p38δ (multiplicity of infection (MOI) 10), and, at 48 h, extracts were prepared for detection of PKCδ, MEP50, H3R8me2s, and H4R3me2s. Similar results were obtained in three different experiments. EV, empty vector. B and C, KERn were infected as above, and, at 48 h, extracts were prepared for ChIP using primers spanning the p21^Cip1 promoter region that includes the p53-2 site. The values are mean ± S.E. (n = 3). *, p < 0.005).

FIGURE 6.

TPA regulates MEP50 and PRMT5 levels and activity. A, KERn were treated with 50 ng/ml TPA for 48 h, and extracts were prepared for detection of MEP50, PRMT5, H3R8me2s, and H4R3me2s. Similar results were obtained in three different experiments. B and C, KERn were treated with 50 ng TPA/ml, and, after 48 h, mRNA extracts were isolated for ChIP analysis and detection of MEP50 and PRMT5 interaction and H3R8-me2s and H4R3me2s formation at the p21^Cip1 promoter. Similar results were obtained in three different experiments. D, cells were treated for 48 h with TPA, and p21^Cip1 levels were monitored.

FIGURE 7.

MEP50 and PRMT5 regulate differentiation and proliferation in an epidermal equivalent model. KERn were electroporated twice with control or MEP50 or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days of exposure at the air-liquid interface, the equivalents were harvested and sectioned. A and B, MEP50 and PRMT5 are required for appropriate skin equivalent formation. KERn were electroporated twice with control, MEP50, or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days of exposure at the air-liquid interface, the equivalents were harvested and stained with hematoxylin and eosin. The nylon support membrane is indicated by an asterisk, and the extent of the epidermis is indicated (E). Similar results were observed in three separate experiments. The graph compares epidermal equivalent thickness among control, MEP50, and PRMT5 siRNA cultures. The values are mean ± S.E. (n = 3). *, p < 0.005. Scale bars = 100 μm. C and D, MEP50 and PRMT5 are required for cell proliferation. KERn were electroporated twice with control, MEP50, or PRMT5 siRNA and seeded for epidermal equivalent culture. After 4 days, the equivalents were stained with Ki67 antibody and Hoechst. The membrane (asterisk) and extent of epidermis (E) are shown. The graph quantitates the number of Ki67-positive cells in the control, MEP50, and PRMT5 siRNA rafts. Similar results were observed in three separate experiments. Significant differences were determined using Student's t test (*, p < 0.005). Note that the blue-green spots are Ki67 staining compared with the larger stained circular structures, which are nonspecific staining to the nylon membrane.

FIGURE 8.

Proposed regulatory model: a balance between transcriptional and epigenetic regulation. Arrows indicate a stimulus, and flat-headed bars inhibition. TPA stimulation or PKCδ overexpression activates MEKK1, MEK3, p38δ, and p53 to increase p21^Cip1 expression, which leads to reduced keratinocyte proliferation. PRMT5/MEP50 arginine dimethylates proteins in the p38δ complex to inhibit p38δ (20), and arginine dimethylates histones H3R8me2s and H4R3me2s in the p21^Cip1 promoter to reduce p21^Cip1 expression, leading to increased cell proliferation. We propose that the PKCδ signaling is dominant in differentiated/non-proliferative cells and that PRMT5-MEP50 is dominant in non-differentiated/proliferative cells. The dashed line indicates that PKCδ/p38δ signaling can reduce PRMT5 and MEP50 expression to suppress pro-proliferation signaling.