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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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DLX3 expression is upregulated by PKCα activation in vivo. (a) Immunohistochemical staining of TPA-treated WT and K5-PKCα transgenic skin with antibodies against KRT5 and DLX3 or KRT5 and Loricrin (LOR). Scale bar, 20 μm. (b) Bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and K5-PKCα skin treated with TPA 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 different areas from three independent mice for each condition. ***P<0.001. (c) Relative expression level of DLX3 and c-Fos in WT or K5-PKCα primary keratinocytes treated with TPA at 1 and 6 h. (d) DLX3 expression level in cultured keratinocytes transduced with Ad-AFOS or Ad-Cntl vectors at 48 h. For all panels: **P<0.01, ***P<0.001 and the results are shown as mean±S.D. of three independent experiments
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fig2: DLX3 expression is upregulated by PKCα activation in vivo. (a) Immunohistochemical staining of TPA-treated WT and K5-PKCα transgenic skin with antibodies against KRT5 and DLX3 or KRT5 and Loricrin (LOR). Scale bar, 20 μm. (b) Bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and K5-PKCα skin treated with TPA 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 different areas from three independent mice for each condition. ***P<0.001. (c) Relative expression level of DLX3 and c-Fos in WT or K5-PKCα primary keratinocytes treated with TPA at 1 and 6 h. (d) DLX3 expression level in cultured keratinocytes transduced with Ad-AFOS or Ad-Cntl vectors at 48 h. For all panels: **P<0.01, ***P<0.001 and the results are shown as mean±S.D. of three independent experiments

Mentions: The biological relevance of the PKCα–DLX3 connection was tested in vivo using an epidermal-targeted, TPA-inducible K5-PKCα transgenic mouse model.20 We found a significant increase in DLX3-expressing cells, as well as higher loricrin expression, in TPA-treated transgenic epidermis (Figures 2a and b). Cultured TPA-treated K5-PKCα keratinocytes displayed DLX3 mRNA induction 6 h after treatment (Figure 2c), which is preceded by the PKC effector c-Fos at 1 h post TPA treatment. The temporal upregulation of c-Fos expression preceding DLX3 was also confirmed by cycloheximide (CHX) treatment of keratinocytes in a time course experiment (Supplementary Figure S3).


A novel DLX3 – PKC integrated signaling network drives keratinocyte differentiation
DLX3 expression is upregulated by PKCα activation in vivo. (a) Immunohistochemical staining of TPA-treated WT and K5-PKCα transgenic skin with antibodies against KRT5 and DLX3 or KRT5 and Loricrin (LOR). Scale bar, 20 μm. (b) Bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and K5-PKCα skin treated with TPA 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 different areas from three independent mice for each condition. ***P<0.001. (c) Relative expression level of DLX3 and c-Fos in WT or K5-PKCα primary keratinocytes treated with TPA at 1 and 6 h. (d) DLX3 expression level in cultured keratinocytes transduced with Ad-AFOS or Ad-Cntl vectors at 48 h. For all panels: **P<0.01, ***P<0.001 and the results are shown as mean±S.D. of three independent experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: DLX3 expression is upregulated by PKCα activation in vivo. (a) Immunohistochemical staining of TPA-treated WT and K5-PKCα transgenic skin with antibodies against KRT5 and DLX3 or KRT5 and Loricrin (LOR). Scale bar, 20 μm. (b) Bar graph showing the number of DLX3-positive cells normalized versus the nuclei in WT and K5-PKCα skin treated with TPA 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 different areas from three independent mice for each condition. ***P<0.001. (c) Relative expression level of DLX3 and c-Fos in WT or K5-PKCα primary keratinocytes treated with TPA at 1 and 6 h. (d) DLX3 expression level in cultured keratinocytes transduced with Ad-AFOS or Ad-Cntl vectors at 48 h. For all panels: **P<0.01, ***P<0.001 and the results are shown as mean±S.D. of three independent experiments
Mentions: The biological relevance of the PKCα–DLX3 connection was tested in vivo using an epidermal-targeted, TPA-inducible K5-PKCα transgenic mouse model.20 We found a significant increase in DLX3-expressing cells, as well as higher loricrin expression, in TPA-treated transgenic epidermis (Figures 2a and b). Cultured TPA-treated K5-PKCα keratinocytes displayed DLX3 mRNA induction 6 h after treatment (Figure 2c), which is preceded by the PKC effector c-Fos at 1 h post TPA treatment. The temporal upregulation of c-Fos expression preceding DLX3 was also confirmed by cycloheximide (CHX) treatment of keratinocytes in a time course experiment (Supplementary Figure S3).

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