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CtBP1 overexpression in keratinocytes perturbs skin homeostasis.

Deng H, Li F, Li H, Deng Y, Liu J, Wang D, Han G, Wang XJ, Zhang Q - J. Invest. Dermatol. (2013)

Bottom Line: Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype.Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription.In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA [2] Department of Pathology, University of Colorado Denver, Aurora, Colorado, USA [3] Department of Dermatology, The Sixth People's Hospital of Shanghai, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT
Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

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Microscopic characteristics of K5.CtBP1 skin. (A) H&E staining of 21 day-old mice shows hyperplasia of epidermis in K5.CtBP1 skin compared with WT skin. (B) Western blotting of CtBP1 in WT and K5.CtBP1 skin. Keratin 14 (K14) was used as loading control. Immunohistochemistry using antibodies specific for CtBP1 (C) and PCNA (D). Scale bar: 40 μm (all panels). qRT-PCR analysis shows down-regulation of CtBP1 target genes p21 (E) and Brca1 (F) in K5.CtBP1 skins. p21 mRNA was decreased to 0.17±0.09 and Brca1 mRNA was decreased to 0.09±0.03 in transgenic skin. (G) Keratinocytes from K5.CtBP1 mice displayed an increased BrdU index. Keratin 14 (red) was used as a counterstain. Scale bar: 15 μm.
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Figure 2: Microscopic characteristics of K5.CtBP1 skin. (A) H&E staining of 21 day-old mice shows hyperplasia of epidermis in K5.CtBP1 skin compared with WT skin. (B) Western blotting of CtBP1 in WT and K5.CtBP1 skin. Keratin 14 (K14) was used as loading control. Immunohistochemistry using antibodies specific for CtBP1 (C) and PCNA (D). Scale bar: 40 μm (all panels). qRT-PCR analysis shows down-regulation of CtBP1 target genes p21 (E) and Brca1 (F) in K5.CtBP1 skins. p21 mRNA was decreased to 0.17±0.09 and Brca1 mRNA was decreased to 0.09±0.03 in transgenic skin. (G) Keratinocytes from K5.CtBP1 mice displayed an increased BrdU index. Keratin 14 (red) was used as a counterstain. Scale bar: 15 μm.

Mentions: Re-activation of CtBP1 expression has been shown in cancer (Deng et al., 2010; Nadauld et al., 2006) and during wound healing (Fig. 1A), presumably contributing to the hyper-proliferative process in these conditions. We biopsied the K5.CtBP1 skin at day 21 postpartum to examine morphology changes (Fig. 2A) and expression of CtBP1 (Fig. 2B and 2C). Hyperplasia in the epidermis of transgenic mice became obvious at the microscopic level (Fig. 2A). Although the K5 promoter targets CtBP1 expression to basal keratinocytes, nuclear CtBP1 staining was apparent throughout the entire epidermis and hair follicles in K5.CtBP1 transgenic mice (Fig. 2C), possibly due to protein retention in differentiated layers. In contrast, weak CtBP1 staining was detected in wildtype skin (Fig. 2B and 2C). Interestingly, although CtBP1 transgene is targeted to keratinocytes, CtBP1-positive cells were also observed in the K5.CtBP1 dermis but not in wildtype dermis (Fig. 2C). This is likely increased endogenous CtBP1 in stromal cells in response to changes in the epidermis and hair follicles. The origin of the cells over-expressing endogenous CtBP1 in the transgenic dermis remains to be determined.


CtBP1 overexpression in keratinocytes perturbs skin homeostasis.

Deng H, Li F, Li H, Deng Y, Liu J, Wang D, Han G, Wang XJ, Zhang Q - J. Invest. Dermatol. (2013)

Microscopic characteristics of K5.CtBP1 skin. (A) H&E staining of 21 day-old mice shows hyperplasia of epidermis in K5.CtBP1 skin compared with WT skin. (B) Western blotting of CtBP1 in WT and K5.CtBP1 skin. Keratin 14 (K14) was used as loading control. Immunohistochemistry using antibodies specific for CtBP1 (C) and PCNA (D). Scale bar: 40 μm (all panels). qRT-PCR analysis shows down-regulation of CtBP1 target genes p21 (E) and Brca1 (F) in K5.CtBP1 skins. p21 mRNA was decreased to 0.17±0.09 and Brca1 mRNA was decreased to 0.09±0.03 in transgenic skin. (G) Keratinocytes from K5.CtBP1 mice displayed an increased BrdU index. Keratin 14 (red) was used as a counterstain. Scale bar: 15 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4537778&req=5

Figure 2: Microscopic characteristics of K5.CtBP1 skin. (A) H&E staining of 21 day-old mice shows hyperplasia of epidermis in K5.CtBP1 skin compared with WT skin. (B) Western blotting of CtBP1 in WT and K5.CtBP1 skin. Keratin 14 (K14) was used as loading control. Immunohistochemistry using antibodies specific for CtBP1 (C) and PCNA (D). Scale bar: 40 μm (all panels). qRT-PCR analysis shows down-regulation of CtBP1 target genes p21 (E) and Brca1 (F) in K5.CtBP1 skins. p21 mRNA was decreased to 0.17±0.09 and Brca1 mRNA was decreased to 0.09±0.03 in transgenic skin. (G) Keratinocytes from K5.CtBP1 mice displayed an increased BrdU index. Keratin 14 (red) was used as a counterstain. Scale bar: 15 μm.
Mentions: Re-activation of CtBP1 expression has been shown in cancer (Deng et al., 2010; Nadauld et al., 2006) and during wound healing (Fig. 1A), presumably contributing to the hyper-proliferative process in these conditions. We biopsied the K5.CtBP1 skin at day 21 postpartum to examine morphology changes (Fig. 2A) and expression of CtBP1 (Fig. 2B and 2C). Hyperplasia in the epidermis of transgenic mice became obvious at the microscopic level (Fig. 2A). Although the K5 promoter targets CtBP1 expression to basal keratinocytes, nuclear CtBP1 staining was apparent throughout the entire epidermis and hair follicles in K5.CtBP1 transgenic mice (Fig. 2C), possibly due to protein retention in differentiated layers. In contrast, weak CtBP1 staining was detected in wildtype skin (Fig. 2B and 2C). Interestingly, although CtBP1 transgene is targeted to keratinocytes, CtBP1-positive cells were also observed in the K5.CtBP1 dermis but not in wildtype dermis (Fig. 2C). This is likely increased endogenous CtBP1 in stromal cells in response to changes in the epidermis and hair follicles. The origin of the cells over-expressing endogenous CtBP1 in the transgenic dermis remains to be determined.

Bottom Line: Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype.Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription.In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA [2] Department of Pathology, University of Colorado Denver, Aurora, Colorado, USA [3] Department of Dermatology, The Sixth People's Hospital of Shanghai, Shanghai Jiaotong University, Shanghai, China.

ABSTRACT
Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

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Related in: MedlinePlus