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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 activation exacerbates DLX3cKO epidermal hyperproliferation. (a) Schematic representation of TPA treatment of WT or DLX3cKO mice. (b) Hematoxylin and eosin staining of TPA-treated WT or DLX3cKO transgenic skin. Scale bar, 20 μm. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice for each condition. ***P<0.001. (c) Immunohistochemical staining of TPA-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 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<0.01. (d) Immunohistochemical staining of TPA-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 +/− TPA. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (e) Immunohistochemical staining of TPA-treated WT and DLX3cKO mice with antibodies against P-PKCα. Nuclei were stained with DAPI. Scale bar, 20 μm
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fig3: PKC activation exacerbates DLX3cKO epidermal hyperproliferation. (a) Schematic representation of TPA treatment of WT or DLX3cKO mice. (b) Hematoxylin and eosin staining of TPA-treated WT or DLX3cKO transgenic skin. Scale bar, 20 μm. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice for each condition. ***P<0.001. (c) Immunohistochemical staining of TPA-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 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<0.01. (d) Immunohistochemical staining of TPA-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 +/− TPA. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (e) Immunohistochemical staining of TPA-treated WT and DLX3cKO mice with antibodies against P-PKCα. Nuclei were stained with DAPI. Scale bar, 20 μm

Mentions: To functionally evaluate the facets of DLX3 and PKCα axis, we used TPA topical application to activate PKC in a mouse model with DLX3 epidermal deletion (K14Cre;DLX3Kin/flox; hereafter referred as DLX3cKO 3) and in WT skin (Figure 3a). TPA treatment led to heightened hyperplasia and hyperkeratosis with significantly increased epidermal thickness and dermal cell infiltration in the DLX3cKO skin when compared with untreated DLX3cKO or TPA-treated WT skin (Figure 3b).


A novel DLX3 – PKC integrated signaling network drives keratinocyte differentiation
PKC activation exacerbates DLX3cKO epidermal hyperproliferation. (a) Schematic representation of TPA treatment of WT or DLX3cKO mice. (b) Hematoxylin and eosin staining of TPA-treated WT or DLX3cKO transgenic skin. Scale bar, 20 μm. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice for each condition. ***P<0.001. (c) Immunohistochemical staining of TPA-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 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<0.01. (d) Immunohistochemical staining of TPA-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 +/− TPA. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (e) Immunohistochemical staining of TPA-treated WT and DLX3cKO mice with antibodies against P-PKCα. Nuclei were stained with DAPI. Scale bar, 20 μm
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fig3: PKC activation exacerbates DLX3cKO epidermal hyperproliferation. (a) Schematic representation of TPA treatment of WT or DLX3cKO mice. (b) Hematoxylin and eosin staining of TPA-treated WT or DLX3cKO transgenic skin. Scale bar, 20 μm. Epidermal thickness was measured and data are presented as mean±S.D. of three different areas from three different mice for each condition. ***P<0.001. (c) Immunohistochemical staining of TPA-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 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<0.01. (d) Immunohistochemical staining of TPA-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 +/− TPA. Data are presented as mean±S.D. of three different areas from three different mice for each condition. (e) Immunohistochemical staining of TPA-treated WT and DLX3cKO mice with antibodies against P-PKCα. Nuclei were stained with DAPI. Scale bar, 20 μm
Mentions: To functionally evaluate the facets of DLX3 and PKCα axis, we used TPA topical application to activate PKC in a mouse model with DLX3 epidermal deletion (K14Cre;DLX3Kin/flox; hereafter referred as DLX3cKO 3) and in WT skin (Figure 3a). TPA treatment led to heightened hyperplasia and hyperkeratosis with significantly increased epidermal thickness and dermal cell infiltration in the DLX3cKO skin when compared with untreated DLX3cKO or TPA-treated WT skin (Figure 3b).

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