Ets1 blocks terminal differentiation of keratinocytes and induces expression of matrix metalloproteases and innate immune mediators

Abstract

The transcription factor Ets1 is normally expressed in the proliferative layer of stratified epithelium, but expression of Ets1 is significantly upregulated in squamous cell carcinomas. How elevated levels of Ets1 impact tumor initiation and progression is not well understood. To determine the biological consequences of overexpression of Ets1, we developed a transgenic mouse model that allows induction of Ets1 expression in keratinocytes of stratified epithelium in a regulatable fashion. Induction of Ets1 during embryonic development results in a dramatic alteration in epidermal structure and function by suppressing the expression of multiple stratum corneum constituents, while at the same time inducing expression of EGF ligands, AP1 transcription factors and matrix metalloproteases. Interestingly, expression of certain immune-related genes, including defensins, chemokines and cytokines was increased as well, suggesting a possible role for immune dysregulation in the promotion of squamous dysplasia. Experiments using cultured mouse keratinocytes indicate that Ets1 can induce expression of some of these mediators in a cell-intrinsic fashion. Collectively, our data reveal that elevated expression of Ets1 has a much broader array of pro-tumorigenic effects on epithelial cells than previously appreciated.

Fig. 1.

Epidermal defects in mice overexpressing Ets1 in the suprabasal layers of epidermis. (A) Expression of the HA-Ets1 transgene in the skin of BT mice. Arrow indicates transgene expression in the inner root sheath of the hair follicle. (B) BT newborn mice are smaller, with an eyes-open-at-birth phenotype (arrow) and have a shiny, translucent skin. (C,D) Hematoxylin and eosin staining to identify epidermal alterations in E18.5 (C) and newborn (NB) (D) BT mice as compared with the wild-type. The BT epidermis is hyperplastic, has a reduced granular layer (white brackets) and is characterized by parakeratosis (arrows) and a compact stratum corneum. Arrowhead in C indicates the presence of a superficial dermal capillary.

Fig. 2.

Epidermal permeability barrier deficiency in mice overexpressing Ets1. (A) Dye exclusion assay on E16.5, E18.5 and newborn (NB) mice. (B) Morphology of newborn wild-type (top) and BT (bottom) cornified envelopes; inset shows higher magnification of the boxed regions. The wild-type demonstrates intact, polygonal cornified envelopes, whereas the BT shows two intact and three fragmented cornified envelopes.

Fig. 3.

Ets1 overexpression in the suprabasal layer blocks keratinocyte terminal differentiation. Immunofluorescent staining of epidermal differentiation markers on dorsal skin of newborn BT and wild-type mice counterstained with β4 integrin to mark the basement membrane. The following markers were used to assess the differentiation pattern of wild-type and BT epidermis: (A) K14, (B) K10, (C) involucrin (INV), (D) filaggrin (FIL) and (E) loricrin (LOR).

Fig. 4.

Ets1 induction in the suprabasal layers results in keratinocyte activation and enhanced proliferation. Immunostaining for keratinocyte activation and proliferation markers on dorsal skin of BT newborn mice and littermate wild-type mice. Samples were counterstained with DAPI to detect nuclei or β4 integrin to mark the basement membrane. The following markers were used (A) K6, (B) ΔNp63, (C) Ki67 and (D) PCNA.

Fig. 5.

Periderm shedding is not affected by suprabasal expression of Ets1. Skin sections of E16.5 embryos stained with (A) hematoxylin and eosin (dashed lines mark the periderm–epidermal junction) or (B) K6, counterstained with DAPI (white dashed line represents the location of epidermal basement membrane, grey dashed line indicates the periderm–epidermal interface). (C,D) Same as above for E18.5 embryonic skin. The arrowheads mark the surface of epidermis.

Fig. 6.

Early hair follicle morphogenesis is similar in wild-type and BT embryos. Skin sections of E18.5 wild-type and BT embryos stained with (A) hematoxylin and eosin or (B) for alkaline phosphatase activity (Alk. Ph.) to mark the dermal papilla (counterstained with hematoxylin). (C) Newborn skin stained with hematoxylin and eosin. (D) Newborn skin stained for alkaline phosphatase and counterstained with DAPI. (E) Newborn skin stained for P-cadherin and counterstained with β4 integrin to mark the basement membrane.

Fig. 7.

Induction of Ets1 leads to major alterations in the differentiation program of keratinocytes. Heatmap visualizations of changes in expression of genes involved in keratinocyte (A) structure, (B) proliferation, (C) motility and (D) immune function. Red indicates increased expression, whereas green indicates decreased expression.

Fig. 8.

Overexpression of Ets1 in cultured keratinocytes drives expression of Mmps and chemokines. (A) Retroviral constructs used to infect keratinocytes: MIGR1, control empty retrovirus; MIGR1-Ets1, retrovirus encoding mouse Ets1; MIGR1-R391D, retrovirus encoding the DNA-binding-defective mutant of Ets1. R391D represents a mutation in a key arginine residue of Ets1 required for DNA binding. This mutant version of Ets1 fails to bind DNA or regulate target genes. (B) RT-PCR analysis of Mmp and chemokine gene expression in retrovirally infected mouse keratinocytes. Ccl2, Ccl20 and Cxcl5 are the three chemokines tested; Mmp9 and Mmp13 are the two Mmps tested; Hprt is an internal control for RNA quantity and integrity.