Interferon regulatory factor 6 is necessary, but not sufficient, for keratinocyte differentiation

Abstract

Regulation of epidermal proliferation and differentiation is critical for maintenance of cutaneous homeostasis. Interferon Regulatory Factor 6 (Irf6)-deficient mice die perinatally and exhibit ectopic proliferation and defective epidermal differentiation. We sought to determine whether these disruptions of epidermal function were cell autonomous, and used embryonic Irf6(-/-) keratinocytes to understand the specific role of Irf6 in keratinocyte proliferation and differentiation. In the absence of Irf6, keratinocytes exhibited a heterogeneous phenotype with the presence of large cells. Irf6(-/-) keratinocytes displayed increased colony-forming efficiency compared with wild-type cells, suggesting that Irf6 represses long-term proliferation. Irf6 was present at low levels in wild-type keratinocytes in culture, and upregulated after induction of differentiation in vitro, along with upregulation of markers of early differentiation. However, Irf6(-/-) keratinocytes did not express markers of terminal differentiation. Overexpression of Irf6 in wild-type keratinocytes was insufficient to induce expression of markers of differentiation under growing conditions. Together, these results indicated that Irf6 is necessary, but not sufficient, for keratinocyte differentiation. Finally, using a transgenic mouse expressing Lac-Z under the regulation of an enhancer element 9.7 kb upstream of the Irf6 start site, we demonstrated that this element contributes to the regulation of Irf6 in the epidermis and keratinocytes in culture.

Figure 1. Characterization of Irf6^−/− keratinocytes

(a) Phase contrast photomicrographs of Irf6^+/+ (left) and Irf6^−/− keratinocytes (right). Black arrows indicate larger keratinocytes in the Irf6^−/− population. (b) Cellular area of wildtype and Irf6^−/− keratinocyte was traced on images as in (a) and averages (N = 6 or 7) plotted. *p < 0.05 after Student t-test. Distribution amongst three arbitrary categories of cellular sizes was calculated, and the difference determined by chi-square contingency test (*p < 0.05). Average of cellular volume (N = 6 or 7) after trypsinization was plotted. (c) Immunofluorescent staining of Krt14 (green, top row) and vimentin (red, top row), Irf6 (red, bottom row) and p63 (green, bottom row). Nuclear DNA is labeled with DAPI (blue). White arrows indicate vimentin-positive, Krt14-negative melanocytes. Scale bar = 100 µm.

Figure 2. Irf6 restricts the long-term proliferative potential of keratinocytes

Percentage of BrdU incorporation is the ratio of BrdU positive cells over the total number of keratinocytes (top left). The distribution of keratinocytes in the cell cycle was determined from DNA content and expressed as percentage of total cells (top right). Number of cells 6 and 10 days after plating were plotted as a function of time (bottom let). Colony forming efficiency was performed and number of colonies (shown by the representative macroscopic view, bottom middle) was counted and percentage of colony forming efficiency calculated for Irf6^+/+ and Irf6^−/− keratinocytes. Data are averages of three to five independent experiments (total N of 4 to 8 per group) ± SEM. *p<0.05; **p < 0.01 after Student t-test.

Figure 3. Irf6 is necessary for terminal differentiation of keratinocytes in vitro

Phase contrast images of Irf6^+/+ (a, b) and Irf6^−/− keratinocytes (c–e) 24 hours in LoCal (a, d) or 72h in KSFM supplemented with 0.15 mM CaCl₂ (K0.15) to induce differentiation (b, c, e). Irf6^−/− keratinocytes were transfected with an adenoviral construct containing Irf6 (AdI) or green fluorescent protein (AdG) cDNA. (f) Western blot analysis of protein extracts from cultured keratinocytes or embryonic skin for Irf6, Krt14, p63, Krt10, Krt1, Involucrin (Inv), loricrin (Lor) and filaggrin (FG; profilaggrin is also detected by this antibody and indicated as ProFG). Molecular weight for each protein is indicated. Black arrow in (f) indicates processed filaggrin in Irf6^+/+ skin; * indicates non-specific band. Scale bar = 100 µm.

Figure 4. Overexpression of Irf6 does not induce keratinocyte differentiation

(a) Microscopic phase contrast images of Irf6^+/+ keratinocytes were transfected with an adenoviral construct (Ad) containing either Irf6 (I) or GFP (G) cDNA. Cultures were then purged of growth factors and grown in KSFM with 0.15 mM CaCl₂ (K0.15), NMedium (N), or NMedium with 0.15 mM CaCl₂ (N0.15) for 72 hours. (b) Proteins were extracted for Western blot analysis and probed for Irf6, Krt14, p63, Krt10, Krt1, loricrin (Lor) and filaggrin (profilaggrin is also detected by this antibody and indicated as ProFG; * indicate non-specific band). Molecular weight for each protein is indicated. Scale bar = 100 µm.

Figure 5. The MCS9.7 enhancer regulates Irf6 in adult epidermis and keratinocytes

a) X-gal staining of MCS9.7-LacZ negative (MCS9.7-LacZ⁻, left) and MCS9.7-LacZ positive (MCS9.7-LacZ⁺) adult tail skin (middle), both counterstained with hematoxylin. Immunofluorescent staining of Irf6 (red) on MCS9.7-LacZ positive adult tail skin (right). Nuclear DNA is labeled with DAPI (blue). (b) X-gal staining of MCS9.7-LacZ negative and MCS9.7-LacZ positive keratinocytes under growing (N0.06) and differentiation conditions (K0.15). (c) Immunofluorescent staining of Irf6 (red) and p63 (green) on MCS9.7-LacZ negative and MCS9.7-LacZ positive keratinocytes under growing and differentiation conditions. Scale bar = 100 µm.