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A novel DLX3 – PKC integrated signaling network drives keratinocyte differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.

No MeSH data available.


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PKC inhibition partially restores epidermal thickness and keratinocyte proliferation in DLX3cKO skin. (a) Schematic representation of GF109203X (GF) treatment of WT or DLX3cKO mice. (b) H&E staining of GF-treated WT and DLX3cKO skin. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice per condition. (c) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against Ki-67. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, Ki-67-positive cell count in WT and DLX3cKO epidermis +/− GF. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (d) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against KRT5 and DLX3. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and DLX3cKO skin treated with GF or acetone (control). DLX3-positive cells and the number of nuclei in each section were determined by ImageJ software analysis of the labeled particles. Data are presented as mean±S.D. of three separate areas from three different mice for each condition. ***P<0.001. (e) Immunohistochemical staining of GF-treated WT and DLX3cKO mice with antibodies against P-PKCα and (f) against KRT5 and KRT10 (upper panel) and Filaggrin (lower panel). Nuclei were stained with DAPI. Scale bar, 20 μm. (g) Total ERK and phosphor-ERK (P-ERK) expression levels determined by western blot of protein extracts from GF-treated WT and DLX3cKO transgenic skin. GAPDH was used as a loading control
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fig5: PKC inhibition partially restores epidermal thickness and keratinocyte proliferation in DLX3cKO skin. (a) Schematic representation of GF109203X (GF) treatment of WT or DLX3cKO mice. (b) H&E staining of GF-treated WT and DLX3cKO skin. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice per condition. (c) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against Ki-67. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, Ki-67-positive cell count in WT and DLX3cKO epidermis +/− GF. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (d) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against KRT5 and DLX3. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and DLX3cKO skin treated with GF or acetone (control). DLX3-positive cells and the number of nuclei in each section were determined by ImageJ software analysis of the labeled particles. Data are presented as mean±S.D. of three separate areas from three different mice for each condition. ***P<0.001. (e) Immunohistochemical staining of GF-treated WT and DLX3cKO mice with antibodies against P-PKCα and (f) against KRT5 and KRT10 (upper panel) and Filaggrin (lower panel). Nuclei were stained with DAPI. Scale bar, 20 μm. (g) Total ERK and phosphor-ERK (P-ERK) expression levels determined by western blot of protein extracts from GF-treated WT and DLX3cKO transgenic skin. GAPDH was used as a loading control

Mentions: Next, we investigated the effect of PKC inhibition in DLX3cKO epidermis by treatment with GF following the same protocol used for TPA treatments (Figure 5a). GF application significantly decreased epidermal thickness compared with the untreated mice (Figure 5b), with decreased hyperplasia and hyperkeratosis and a reduction of Ki-67-positive cells (Figure 5c). A reduced number of DLX3-positive cells in GF-treated WT skin was detected (Figure 5d). The epidermal hyperplasia in DLX3cKO skin was also normalized by the specific PKCα inhibitor Gö6976 (Supplementary Figure S5).


A novel DLX3 – PKC integrated signaling network drives keratinocyte differentiation
PKC inhibition partially restores epidermal thickness and keratinocyte proliferation in DLX3cKO skin. (a) Schematic representation of GF109203X (GF) treatment of WT or DLX3cKO mice. (b) H&E staining of GF-treated WT and DLX3cKO skin. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice per condition. (c) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against Ki-67. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, Ki-67-positive cell count in WT and DLX3cKO epidermis +/− GF. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (d) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against KRT5 and DLX3. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and DLX3cKO skin treated with GF or acetone (control). DLX3-positive cells and the number of nuclei in each section were determined by ImageJ software analysis of the labeled particles. Data are presented as mean±S.D. of three separate areas from three different mice for each condition. ***P<0.001. (e) Immunohistochemical staining of GF-treated WT and DLX3cKO mice with antibodies against P-PKCα and (f) against KRT5 and KRT10 (upper panel) and Filaggrin (lower panel). Nuclei were stained with DAPI. Scale bar, 20 μm. (g) Total ERK and phosphor-ERK (P-ERK) expression levels determined by western blot of protein extracts from GF-treated WT and DLX3cKO transgenic skin. GAPDH was used as a loading control
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fig5: PKC inhibition partially restores epidermal thickness and keratinocyte proliferation in DLX3cKO skin. (a) Schematic representation of GF109203X (GF) treatment of WT or DLX3cKO mice. (b) H&E staining of GF-treated WT and DLX3cKO skin. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice per condition. (c) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against Ki-67. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, Ki-67-positive cell count in WT and DLX3cKO epidermis +/− GF. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (d) Immunohistochemical staining of GF-treated WT and DLX3cKO transgenic skin with antibodies against KRT5 and DLX3. Nuclei were stained with DAPI. Scale bar, 20 μm. Bottom panel, bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and DLX3cKO skin treated with GF or acetone (control). DLX3-positive cells and the number of nuclei in each section were determined by ImageJ software analysis of the labeled particles. Data are presented as mean±S.D. of three separate areas from three different mice for each condition. ***P<0.001. (e) Immunohistochemical staining of GF-treated WT and DLX3cKO mice with antibodies against P-PKCα and (f) against KRT5 and KRT10 (upper panel) and Filaggrin (lower panel). Nuclei were stained with DAPI. Scale bar, 20 μm. (g) Total ERK and phosphor-ERK (P-ERK) expression levels determined by western blot of protein extracts from GF-treated WT and DLX3cKO transgenic skin. GAPDH was used as a loading control
Mentions: Next, we investigated the effect of PKC inhibition in DLX3cKO epidermis by treatment with GF following the same protocol used for TPA treatments (Figure 5a). GF application significantly decreased epidermal thickness compared with the untreated mice (Figure 5b), with decreased hyperplasia and hyperkeratosis and a reduction of Ki-67-positive cells (Figure 5c). A reduced number of DLX3-positive cells in GF-treated WT skin was detected (Figure 5d). The epidermal hyperplasia in DLX3cKO skin was also normalized by the specific PKCα inhibitor Gö6976 (Supplementary Figure S5).

View Article: PubMed Central - PubMed

ABSTRACT

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C &alpha; (PKC&alpha;) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKC&alpha; by transgenic targeting (K5-PKC&alpha;), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKC&alpha; activation, suggesting a feedback regulation of DLX3 and PKC&alpha;. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3&ndash;PKC&alpha; signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.

No MeSH data available.


Related in: MedlinePlus