Selective Modulation of Hedgehog/GLI Target Gene Expression by Epidermal Growth Factor Signaling in Human Keratinocytes
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In mammalian skin, HH/GLI signaling plays a prominent role in the development of epidermal appendages and skin cancer.
Loss of HH signaling in murine epidermis results in an arrest of hair follicle growth at an early stage of follicle development due to reduced proliferation...
while constitutive pathway activation in epidermal cells induces (with high frequency) tumors with basal cell carcinoma
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IL1R2, EGFR, and p-ERK are coexpressed in the ORS of human hair follicles. To assess the in vivo relevance of our data on EGF-dependent modulation of GLI target gene specificity, we analyzed the protein expression patterns of EGFR, active ERK1/2 (p-ERK), and the GLI/EGF target IL1R2 in human skin (Fig. 6). GLI1 and GLI2 have previously been shown to be expressed in the outer root sheath (ORS) of human anagen hair follicles (25, 38). Consistent with our in vitro data, we found strong expression of IL1R2 in the ORS of anagen hair follicles, where staining was restricted to the region below the sebaceous gland (Fig. 6A and C). A similar pattern was observed for EGFR, though staining extended further towards the hair bulb (Fig. 6B and D). Double immunofluorescence analysis clearly revealed coexpression of IL1R2 (Fig. 6E) and EGFR (Fig. 6F) in ORS cells. In line with the activation of IL1R2 expression by synergistic GLI and EGFR/ERK signaling, phosphorylated nuclear p-ERK1/2 protein also localized to the ORS (Fig. 6G). Together with the findings that the JAG2 and CCND1 GLI/EGF target genes are also expressed in the ORS region of anagen hair follicles (74, 107), the results suggest that synergistic signaling of the EGFR and HH/GLI signal transduction pathways shown in vitro may also operate in vivo. However, the data do not rule out the possibility that ERK1/2 may also be activated by signaling pathways other than EGFR.
Modulation of epidermal stem cell marker expression and keratinocyte proliferation by GLI1/EGF signaling. The expression patterns of IL1R2 and EGFR in a region of the ORS that lies in or close to the putative stem cell niche (bulge region) (23) of human hair follicles may point to a role of combined HH/GLI and EGFR signaling in modulating the fate of ORS cells. We addressed this speculation by analyzing the expression of hair follicle stem cell markers (KRT15, KRT19, NES, TNC, and CD71) (52, 60, 96, 103) in human N/TERT-1 keratinocytes in response to GLI1 and EGFR signaling. As shown in Fig. 7A, expression of GLI1 in the absence of extracellular stimuli induced elevated mRNA levels of all stem cell markers tested. Intriguingly, the addition of EGF essentially neutralized the effect of GLI1 such that expression levels of stem cell markers were comparable to control cells transduced with EGFP-expressing retrovirus. Accordingly, mRNA levels of the CD71 gene, which encodes the transferrin receptor that is absent in stem cells (96), increased in response to GLI1/EGF. These results show that GLI1 can elicit a stem cell-like expression signature in epidermal cells and that this signature can be prevented by simultaneous EGF signaling.
Loss of stem cell-like expression signatures by treatment of GLI1 keratinocytes with EGF may point to a proliferative role of combinatorial GLI/EGF signaling in human skin. This is supported by the observation that both HH/GLI and EGFR signaling have previously been implicated in proliferative control and growth of human keratinocytes and anagen hair follicles, respectively, and by our own data presented in this study showing enhanced induction of CCND1 expression by concomitant GLI1 and EGF signaling (16, 28, 58, 67, 72, 94, 95). Furthermore, we have previously shown that GLI1 and GLI2 promote G1-phase-to-S-phase progression in serum-containing medium, while they fail to do so under starving conditions, suggesting that additional mitogenic stimuli are required to allow GLI proteins to exert their proliferative activity (75, 77) (G. Regl and F. Aberger, unpublished data). We therefore asked whether the proliferative effect of GLI1 on human keratinocytes depends on parallel EGFR signaling. As shown in Fig. 7B and C, expression of tetracycline-inducible GLI1 in confluent HaCaT keratinocyte cultures induced entry into S phase as monitored by BrdU incorporation. EGF alone or in combination with GLI1 did not further increase the number of cells in S phase. However, presence of the selective EGFR-inhibitor gefitinib (0.5 μM) completely abrogated the S-phase-promoting effect of GLI1, suggesting that activation of EGFR signaling is essential for GLI-induced proliferation. At this concentration (and even up to 1.5 μM), gefitinib affects neither the expression of EGF-independent GLI targets, such as PTCH and BCL2 genes, nor the overall cell viability (also see Fig. 4A) (data not shown); concentrations higher than 1.5 μM were not tested. qRT-PCR analysis of cell cycle progression genes revealed, to a variable extent, enhanced expression in response to combinatorial GLI1/EGF signaling compared to either stimulus alone. Consistent with results obtained with BrdU incorporation assays, the addition of 0.5 μM gefitinib inhibited GLI1- and GLI1/EGF-induced expression of cell cycle progression genes, most notably expression of CKS1B and CDC45L (Fig. 7D). The results uncover an essential role for EGFR signaling in GLI-induced cell cycle progression and suggest that combinatorial HH/GLI and EGFR signaling promote keratinocyte proliferation by cooperative induction of cell cycle progression genes.
