Differential regulation of epidermal function by VDR coactivators

Abstract

The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes. These coactivator complexes are not interchangeable in their regulation of keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Moreover, we have found a specific role for DRIP205 in the regulation of beta-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion.

Figure 1. A model showing the differential expression of various markers and functions in the epidermis with differentiation

DRIP205 is most highly expressed in the stratum basale and spinosum where it participates with VDR in regulating proliferation. SRC3 on the other hand is found in highest concentration in the stratum granulosum where it participates with VDR in the regulation of terminal differentiation including formation of the permeability barrier and innate immune responsiveness.

Figure 2. Impact of VDR, DRIP205, and SRC3 knockdown on keratinocyte morphology, proliferation and apoptosis

The keratinocytes were transfected with siRNAs to VDR, DRIP205, and SRC3, respectively. The efficiency and specificity of the knockdowns are shown at both the mRNA (A) and protein (B) level. Phase contrast was used to assess morphology (C). Proliferation was evaluated using BrdU (C,D), and apoptosis was determined by TUNEL staining (E). Statistical significance is shown by asterisks (*P<0.05 **P<0.01).

Figure 3. Regulation by VDR and DRIP205 of cyclin D1 and Gli1 expression

The keratinocytes were transfected with the specific and control siRNAs to VDR, DRIP205, and SRC3. The cells were treated with 10nM 1,25(OH)₂D₃ or vehicle as in Fig. 2. The mRNA levels of cyclin D1, Gli1, and control L19 were measured by real time PCR. The expression is shown as % of the sicontrol vehicle cultures. The error bars enclose mean +/− SD of triplicate cultures. Statistical significance is shown by asterisks (*P<0.05 **P<0.01).

Figure 4. Impact of DRIP205, SRC3, and VDR silencing on calcium induced expression of keratinocyte differentiation markers

The keratinocytes were transfected with siRNAs to VDR, DRIP205, and SRC3, respectively. The cells were then switched to 1.2mM calcium, and the mRNA levels of the indicated differentiation markers determined at 3 and 7 days by qRTPCR. Silencing of both DRIP205 and SRC3, as well as VDR inhibited the calcium induced expression of these markers. The error bars enclose mean +/−SD of triplicate cultures, and in most cases are within the symbol.

Figure 5. VDR and SRC3 but not DRIP205 regulate production of epidermal specific GlcCer species, the formation of lamellar bodies, and the key enzymes and transporter involved

Human epidermal keratinocytes were infected with shRNA adenovirus for VDR and its coactivators to block their expression. Keratinocytes were maintained in medium supplemented with vitamin C and serum to induce LB production. A. The levels of key lipids were determined expressed as % of control. Data are represented as mean +/− SD of three measurements. (* p<0.03). B. The tissues were examined by electron microscopy to determine number of LBs and their content (left panels). The lipid secretion and processing were evaluated using ruthenium staining (right panels). Bars represent 1.0 μm (left panels) and 0.2 μm (right panels). The table summarizes the results. C. The transcription of lipid synthesis enzymes was evaluated by qRTPCR. The mRNA levels were normalized to control gene L19, and relative expression to control group was expressed as a percentage (%). Only the significantly affected genes are shown (ELOVL4, UGCG, and ABCA12). Data are represented as mean +/− SD of at least three independent experiments (* p<0.01).

Figure 6. The innate immune response in the epidermis requires 1,25(OH)₂D₃ and the coactivator SRC3

A. The expression of cathelicidin, CD14, CYP27b1, and TLR2 (but not other TLRs) in human skin were increased by wounding. B. This experiment was repeated in CYP27b1 null mice demonstrating that CYP27b1 was required for the induction of CD14 and cathelicidin. C. Cultured human keratinocytes were used to demonstrate the ability of 1,25(OH)₂D₃ (100nM) to stimulate TLR2, CD14, and cathelicidin. D. Silencing of SRC3 but not of DRIP205 blocked the ability of 1,25(OH)₂D₃ to induce cathelicidin expression in these cultured human keratinocytes. All data are expressed as mean +/− SD of triplicate experiments.