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121 The role of Runx family in the murine epidermis
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Leucine-rich alpha-2-glycoprotein 1, which is down-regulated during skin aging, plays a role in the maintenance of extracellular matrix integrity S Lee1, S Ahn1, Y Lee1, D Lee2 and J Chung2 1 Biochemistry, Yonsei University, Seoul, Korea (the Republic of) and 2 Dermatology, Seoul National University, Seoul, Korea (the Republic of) The skin aging process can be divided into intrinsic aging and photo-aging. Photo-aging has been studied extensively, but intrinsic aging is poorly understood. In an attempt to identify genes involved in the intrinsic skin aging, we analyzed differentially expressed genes (DEGs) by RNA-sequencing analysis of dorsal skin tissues from 3-month-old (young) and 24-monthold (old) SKH1 hairless mice. Transcriptome data showed that over 1500 genes are upregulated or down-regulated during intrinsic skin aging. The transcript of the gene encoding leucine-rich alpha-2-glycoprotein 1 (LRG1) was significantly down-regulated in skin tissues of the old group. Although LRG1 is known as a positive regulator of transforming growth factorbeta signaling pathway, its function and the detailed mechanism in the skin aging have not been well investigated. Our results showed that mRNA and protein levels of LRG1 significantly decreased in the skin of the old group compared with that of the young group. A recombinant human LRG1 (rhLRG1) was expressed and purified from HEK 293 cells. Treatment of rhLRG1 increased the synthesis of type I collagen and reduced the secretion of MMP-1 in human dermal fibroblasts. In addition, rhLRG1 increased the phosphorylation of Smads and an inhibitor of type I TGF-beta receptor abolished the rhLRG1-induced phosphorylation of Smads, suggesting that LRG1 activates the TGF-beta signaling pathway. In these regards, our results show that LRG1 has a potential to retard skin aging by inducing TGF-beta signaling pathway and then maintaining extracellular matrix integrity.
Study on skin barrier function in cutaneous field cancerization in a murine model J Santiago1, JM Pe´rez-Ortiz1, J Mun˜oz-Rodrı´guez1, M de la Cruz-Morcillo1, C Villar1, A Gonza´lez-Lo´pez1, N Gallardo2, V Lo´pez Go´mez-Carren˜o2, F Redondo-Calvo1 and E Gala´nMoya2 1 Translational Research Unit-Dermatology service, University General Hospital, Ciudad Real, Spain and 2 Regional Center for Biomedical Research, University of Castilla-La Mancha, Ciudad Real/Albacete, Spain In cutaneous field cancerization (CFC), stem cells accumulate genetic mutations following chronic UV light exposure and then expand to create an area of premalignant cells, subclinical and clinical actinic keratoses (AKs), and squamous cell carcinomas (SCCs). Ageing and UV light are associated with structural and functional changes of the stratum corneum (SC) that result in impaired skin barrier, but few is known about this process in chronic actinic damage. The aim is to check if skin barrier is impaired in CFC respect to non-exposed skin. Skh1 male mice (n ¼ 5), 8 months old, were exposed to UVB (l ¼ 302 nm) at 150 mJ/cm2 once daily for 3 months, and compared to a control group (non-exposed to UVB). Skin samples were collected to perform: histopathology, Nile Red staining, immunohistochemistry, cholesterol quantification, and qPCR for genes related to SC physiology. Skin barrier parameters (TEWL, surface pH, and SC hydration) were severely affected in CFC respect to nonexposed mice. Filaggrin, involucrin, locricrin, total cholesterol, enzymes related to epidermal lipid synthesis and lipid transporter ABCA12 were increased in CFC murine model. Our findings show an impaired skin barrier in CFC respect to normal skin. Chronic exposure to UVB light induces hyperproliferation of keratinocytes, disorganization of SC, and inflammation, which characterize epidermal changes in CFC. Although keratinocytes increase the expression of structural proteins of SC, together with lipid synthesis and secretion, the epidermal architecture is dramatically distorted. Thus, skin barrier function is severly impaired in CFC and might also promote hyperproliferation of epidermal stem cells, facilitating clonal expansion of premalignant cells.
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POMP increases proteasome assembly and activity in psoriatic lesional skin B Zieba2, L Henry2, M Lacroix4, T Lavabre-Bertrand2, L Meunier1,2, O Coux3 and P Stoebner1,2 1 Dermatology, CHU Nıˆmes, Nıˆmes, France, 2 CNRS UMR5247, Montpellier, France, 3 CNRS UMR5237, Montpellier, France and 4 INSERM U1194, Montpellier, France The ubiquitin-proteasome pathway is involved in the pathogenesis of psoriasis and proteasome subunits are increased in lesional psoriatic skin. Recent works have highlighted that proteasome levels can be regulated through modulation of proteasome assembly notably by the proteasome maturation protein POMP. We investigated whether proteasome assembly and POMP expression are modified in psoriatic skin. Proteasome assembly as well as expression of proteasome regulators were assessed in non-lesional and lesional psoriatic skin using native gel electrophoresis and western blots, respectively. The protein and mRNA expression levels of POMP were compared by western blots, immunohistochemistry and qPCR. The role of POMP in keratinocyte proliferation and differentiation was assessed by silencing POMP gene expression by RNA interference in HaCaT keratinocytes. We show that both 20S and 26S proteasomes (and their respective proteolytic activities) as well as the main proteasome regulators (PA28ab, PA28g, PA200 and 19S complex) are increased in lesional psoriatic skin. POMP binds to 20S precursor complexes and is overexpressed in lesional epidermal psoriatic skin, supporting that POMP-mediated proteasome assembly is increased in psoriatic skin. POMP silencing inhibited HaCaT cell proliferation and induced apoptosis through the inhibition of proteasome assembly. Moreover POMP partial depletion decreased the expression of the differentiation markers keratin 10 and involucrin during the calcium induced HaCaT cells differentiation. Altogether these results suggest a role for POMP and proteasome assembly in psoriasis pathogenesis.
Changes in fatty acid lengths of ceramides toward shorter chain dominance in human psoriasis skin B Kim1, J Shon2, K Liu2, S Hong3 and S Ahn1 1 Department of Dermatology, Yonsei University Wonju College of Medicine, Wonju, Korea (the Republic of), 2 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea (the Republic of) and 3 Department of Dermatology, College of Medicine, Dankook University, Cheon-An, Korea (the Republic of) Ceramides(CERs) are major component of the intercellular lipids, and average fatty acid(FA) chain length of the CERs correlate with the barrier function. Changes in skin lipid profiles have been associated with abnormal barrier function permeability in psoriasis. However, it is unknown how lipid synthetic enzymes affect the synthesis of ceramides. Here, we aimed to identify the alteration of FA chain length of CERs and lipid synthetic enzymes including elongases(ELOVLs) in human psoriasis skin. Stratum corneum(SC) and epidermal tissues were collected from 12 subjects with psoriasis and 12 controls. To examine the alteration of FA lengths of CERs, the each species of CERs in SCs were quantified using LC-MS. We found that the profiles of FA lengths of CERs shifted toward shorter chain in SC of psoriasis lesions. The unbound CERs ([NH],[NP]) with very long FAs(longer than C26) decreased whereas those with shorter FAs(shorter than C24) increased in psoriasis skin. To evaluate the changes in lipid enzymes affecting the FA chain lengths, the mRNA expression of elongase(ELOVL) 1 and ceramide synthase(CerS)3 in the epidermis was evaluated by using quantitative RTePCR. In psoriasis epidermis, ELOVL1 were downregulated whereas CerS3 showed no significant difference compared with controls. These enzymatic changes may lead to impairment in synthesis of CERs with very long FAs and then tilt the balance toward the dominance of CERs with shorter FAs. Thus, these findings suggest that the profile changes of FA lengths of CERs mediated by enzymatic changes, especially ELOVLs, can be associated in part with barrier dysfunction in psoriasis.
The role of Runx family in the murine epidermis E Ogawa, T Edamitsu and R Okuyama Dermatology, Shinshu university school of medicine, Matsumoto, Japan Keratinocytes contribute to the epidermal homeostasis mainly. Keratinocytes regularly undergo differentiation and proliferation so that the homeostasis is maintained normally. Many factor are involved in these regulation. However, the regulatory mechanism of keratinocytes is complicated and its full picture has not been found out. Runt-related transcription factor (RUNX) proteins belong to a family of metazoan transcription factors that serve as master regulators. The RUNX gene family has three members, Runx1(R1), Runx2 and Runx3(R3). RUNX functions have been unclear in keratinocytes in detail. In this study, we focused on the roles of RUNX in keratinocyte. First, we confirmed whether R1 and R3 (R1/3) are expressed in keratinocytes. We used primary culture of mouse keratinocytes. The transcripts of R1/3 were detected by RT-PCR. Keratinocytes expressed R1/3. Next, we overexpressed R1/3 in primary mouse keratinocytes. R1/3 overexpression decreased the amounts of keratin1 and keratin10 (K1/10). Conversely, R1/3 knockdown increased the amounts of K1/10. R1/3 inhibited the induction of K1/10, which were early differentiation markers. We analyzed the mechanism to modulate K1/10 by R1/3. We tried to immunostain R1/3. Immunohistological analysis showed that R1/3 were detected in the nuclear of immatured keratinocytes but moved out of the nuclear of differentiated keratinocytes. ChIP assay showed that R1/3 bound to Runx consensus sequences of K1/10 genome in undifferentiated state but not in differentiated state. R1/3 directly regulated the expressions of K1/10. We assessed the role of R1/3 to proliferation. R1/3 overexpression induced keratinocytes growth arrest. R1/3 knockdown did not advance proliferation. We generated transgenic mice lacking R1(or R3) in basal kerationocytes. In these mice, K1/10 were detected in basal keratinocytes, but the proliferation did not changed. Last we applied Imiquimod to these mice. However there was no difference between transgenic mice and wild type. Taken together, we propose that R1/3 regulate partially differentiation and proliferation of keratinocytes.
The fatty-acid chain length of ceramides is negatively affected by chronic UV exposure M Park1, H Seo1,4, B Kim2, J Son3, K Liu3, B Park1, M Kim1 and S Hong1,4 1 Department of Dermatology, College of Medicine, Dankook University, Cheonan, Korea (the Republic of), 2 Department of Dermatology, Yonsei University Wonju College of Medicine, Wonju, Korea (the Republic of), 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea (the Republic of) and 4 Wellskin Research Center, Dankook University Hospital, Cheonan, Korea (the Republic of) The proportions of ceramides with very long-chain fatty acids (FAs) has been known to be reduced in the stratum corneum of patients with atopic dermatitis and psoriasis. It could negatively affects the proper function of skin barrier as well. Meanwhile, it has been reported that photoaged murine skin displayed abnormal barrier function and decreased lipid levels of stratum corneum. So, to further know the alteration of FA chain length of ceramides and lipid synthesis-related enzymes after long-term UV exposure, the profile of ceramide chain length was analyzed in photoaged mouse epidermis. Compared to age-matched untreated skin, some of unbound ceramides with longer than 26 carbons (such as ceramide NP and NDS) were significantly decreased, but bound ceramides (such as ceramide OS) only showed a decreasing tendency. There was a significantly decrease in mRNA expression of FA elongase ELOVL1 and ceramide synthase 3 in the photoaged murine epidermis. These enzymes are essentially involved in the formation of long-chain ceramides. To investigate the in vitro effect of low dose repeated UV exposure, the expression of lipid synthetic enzymes and their known transcription factors (such as PPARs and LXRa) was assessed after 6 times of low dose (5mJ/ cm2) of UVB radiation exposures to human epidermal keratinocyte. The expression of LXRa and PPARg was significantly suppressed in this in vitro photoaging model. Our results demonstrate ceramide chain length may be negatively affected in photoaged skin, as like atopic dermatitis.
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Leucine-rich alpha-2-glycoprotein 1, which is down-regulated during skin aging, plays a role in the maintenance of extracellular matrix integrity S Lee1, S Ahn1, Y Lee1, D Lee2 and J Chung2 1 Biochemistry, Yonsei University, Seoul, Korea (the Republic of) and 2 Dermatology, Seoul National University, Seoul, Korea (the Republic of) The skin aging process can be divided into intrinsic aging and photo-aging. Photo-aging has been studied extensively, but intrinsic aging is poorly understood. In an attempt to identify genes involved in the intrinsic skin aging, we analyzed differentially expressed genes (DEGs) by RNA-sequencing analysis of dorsal skin tissues from 3-month-old (young) and 24-monthold (old) SKH1 hairless mice. Transcriptome data showed that over 1500 genes are upregulated or down-regulated during intrinsic skin aging. The transcript of the gene encoding leucine-rich alpha-2-glycoprotein 1 (LRG1) was significantly down-regulated in skin tissues of the old group. Although LRG1 is known as a positive regulator of transforming growth factorbeta signaling pathway, its function and the detailed mechanism in the skin aging have not been well investigated. Our results showed that mRNA and protein levels of LRG1 significantly decreased in the skin of the old group compared with that of the young group. A recombinant human LRG1 (rhLRG1) was expressed and purified from HEK 293 cells. Treatment of rhLRG1 increased the synthesis of type I collagen and reduced the secretion of MMP-1 in human dermal fibroblasts. In addition, rhLRG1 increased the phosphorylation of Smads and an inhibitor of type I TGF-beta receptor abolished the rhLRG1-induced phosphorylation of Smads, suggesting that LRG1 activates the TGF-beta signaling pathway. In these regards, our results show that LRG1 has a potential to retard skin aging by inducing TGF-beta signaling pathway and then maintaining extracellular matrix integrity.
Study on skin barrier function in cutaneous field cancerization in a murine model J Santiago1, JM Pe´rez-Ortiz1, J Mun˜oz-Rodrı´guez1, M de la Cruz-Morcillo1, C Villar1, A Gonza´lez-Lo´pez1, N Gallardo2, V Lo´pez Go´mez-Carren˜o2, F Redondo-Calvo1 and E Gala´nMoya2 1 Translational Research Unit-Dermatology service, University General Hospital, Ciudad Real, Spain and 2 Regional Center for Biomedical Research, University of Castilla-La Mancha, Ciudad Real/Albacete, Spain In cutaneous field cancerization (CFC), stem cells accumulate genetic mutations following chronic UV light exposure and then expand to create an area of premalignant cells, subclinical and clinical actinic keratoses (AKs), and squamous cell carcinomas (SCCs). Ageing and UV light are associated with structural and functional changes of the stratum corneum (SC) that result in impaired skin barrier, but few is known about this process in chronic actinic damage. The aim is to check if skin barrier is impaired in CFC respect to non-exposed skin. Skh1 male mice (n ¼ 5), 8 months old, were exposed to UVB (l ¼ 302 nm) at 150 mJ/cm2 once daily for 3 months, and compared to a control group (non-exposed to UVB). Skin samples were collected to perform: histopathology, Nile Red staining, immunohistochemistry, cholesterol quantification, and qPCR for genes related to SC physiology. Skin barrier parameters (TEWL, surface pH, and SC hydration) were severely affected in CFC respect to nonexposed mice. Filaggrin, involucrin, locricrin, total cholesterol, enzymes related to epidermal lipid synthesis and lipid transporter ABCA12 were increased in CFC murine model. Our findings show an impaired skin barrier in CFC respect to normal skin. Chronic exposure to UVB light induces hyperproliferation of keratinocytes, disorganization of SC, and inflammation, which characterize epidermal changes in CFC. Although keratinocytes increase the expression of structural proteins of SC, together with lipid synthesis and secretion, the epidermal architecture is dramatically distorted. Thus, skin barrier function is severly impaired in CFC and might also promote hyperproliferation of epidermal stem cells, facilitating clonal expansion of premalignant cells.
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POMP increases proteasome assembly and activity in psoriatic lesional skin B Zieba2, L Henry2, M Lacroix4, T Lavabre-Bertrand2, L Meunier1,2, O Coux3 and P Stoebner1,2 1 Dermatology, CHU Nıˆmes, Nıˆmes, France, 2 CNRS UMR5247, Montpellier, France, 3 CNRS UMR5237, Montpellier, France and 4 INSERM U1194, Montpellier, France The ubiquitin-proteasome pathway is involved in the pathogenesis of psoriasis and proteasome subunits are increased in lesional psoriatic skin. Recent works have highlighted that proteasome levels can be regulated through modulation of proteasome assembly notably by the proteasome maturation protein POMP. We investigated whether proteasome assembly and POMP expression are modified in psoriatic skin. Proteasome assembly as well as expression of proteasome regulators were assessed in non-lesional and lesional psoriatic skin using native gel electrophoresis and western blots, respectively. The protein and mRNA expression levels of POMP were compared by western blots, immunohistochemistry and qPCR. The role of POMP in keratinocyte proliferation and differentiation was assessed by silencing POMP gene expression by RNA interference in HaCaT keratinocytes. We show that both 20S and 26S proteasomes (and their respective proteolytic activities) as well as the main proteasome regulators (PA28ab, PA28g, PA200 and 19S complex) are increased in lesional psoriatic skin. POMP binds to 20S precursor complexes and is overexpressed in lesional epidermal psoriatic skin, supporting that POMP-mediated proteasome assembly is increased in psoriatic skin. POMP silencing inhibited HaCaT cell proliferation and induced apoptosis through the inhibition of proteasome assembly. Moreover POMP partial depletion decreased the expression of the differentiation markers keratin 10 and involucrin during the calcium induced HaCaT cells differentiation. Altogether these results suggest a role for POMP and proteasome assembly in psoriasis pathogenesis.
Changes in fatty acid lengths of ceramides toward shorter chain dominance in human psoriasis skin B Kim1, J Shon2, K Liu2, S Hong3 and S Ahn1 1 Department of Dermatology, Yonsei University Wonju College of Medicine, Wonju, Korea (the Republic of), 2 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea (the Republic of) and 3 Department of Dermatology, College of Medicine, Dankook University, Cheon-An, Korea (the Republic of) Ceramides(CERs) are major component of the intercellular lipids, and average fatty acid(FA) chain length of the CERs correlate with the barrier function. Changes in skin lipid profiles have been associated with abnormal barrier function permeability in psoriasis. However, it is unknown how lipid synthetic enzymes affect the synthesis of ceramides. Here, we aimed to identify the alteration of FA chain length of CERs and lipid synthetic enzymes including elongases(ELOVLs) in human psoriasis skin. Stratum corneum(SC) and epidermal tissues were collected from 12 subjects with psoriasis and 12 controls. To examine the alteration of FA lengths of CERs, the each species of CERs in SCs were quantified using LC-MS. We found that the profiles of FA lengths of CERs shifted toward shorter chain in SC of psoriasis lesions. The unbound CERs ([NH],[NP]) with very long FAs(longer than C26) decreased whereas those with shorter FAs(shorter than C24) increased in psoriasis skin. To evaluate the changes in lipid enzymes affecting the FA chain lengths, the mRNA expression of elongase(ELOVL) 1 and ceramide synthase(CerS)3 in the epidermis was evaluated by using quantitative RTePCR. In psoriasis epidermis, ELOVL1 were downregulated whereas CerS3 showed no significant difference compared with controls. These enzymatic changes may lead to impairment in synthesis of CERs with very long FAs and then tilt the balance toward the dominance of CERs with shorter FAs. Thus, these findings suggest that the profile changes of FA lengths of CERs mediated by enzymatic changes, especially ELOVLs, can be associated in part with barrier dysfunction in psoriasis.
The role of Runx family in the murine epidermis E Ogawa, T Edamitsu and R Okuyama Dermatology, Shinshu university school of medicine, Matsumoto, Japan Keratinocytes contribute to the epidermal homeostasis mainly. Keratinocytes regularly undergo differentiation and proliferation so that the homeostasis is maintained normally. Many factor are involved in these regulation. However, the regulatory mechanism of keratinocytes is complicated and its full picture has not been found out. Runt-related transcription factor (RUNX) proteins belong to a family of metazoan transcription factors that serve as master regulators. The RUNX gene family has three members, Runx1(R1), Runx2 and Runx3(R3). RUNX functions have been unclear in keratinocytes in detail. In this study, we focused on the roles of RUNX in keratinocyte. First, we confirmed whether R1 and R3 (R1/3) are expressed in keratinocytes. We used primary culture of mouse keratinocytes. The transcripts of R1/3 were detected by RT-PCR. Keratinocytes expressed R1/3. Next, we overexpressed R1/3 in primary mouse keratinocytes. R1/3 overexpression decreased the amounts of keratin1 and keratin10 (K1/10). Conversely, R1/3 knockdown increased the amounts of K1/10. R1/3 inhibited the induction of K1/10, which were early differentiation markers. We analyzed the mechanism to modulate K1/10 by R1/3. We tried to immunostain R1/3. Immunohistological analysis showed that R1/3 were detected in the nuclear of immatured keratinocytes but moved out of the nuclear of differentiated keratinocytes. ChIP assay showed that R1/3 bound to Runx consensus sequences of K1/10 genome in undifferentiated state but not in differentiated state. R1/3 directly regulated the expressions of K1/10. We assessed the role of R1/3 to proliferation. R1/3 overexpression induced keratinocytes growth arrest. R1/3 knockdown did not advance proliferation. We generated transgenic mice lacking R1(or R3) in basal kerationocytes. In these mice, K1/10 were detected in basal keratinocytes, but the proliferation did not changed. Last we applied Imiquimod to these mice. However there was no difference between transgenic mice and wild type. Taken together, we propose that R1/3 regulate partially differentiation and proliferation of keratinocytes.
The fatty-acid chain length of ceramides is negatively affected by chronic UV exposure M Park1, H Seo1,4, B Kim2, J Son3, K Liu3, B Park1, M Kim1 and S Hong1,4 1 Department of Dermatology, College of Medicine, Dankook University, Cheonan, Korea (the Republic of), 2 Department of Dermatology, Yonsei University Wonju College of Medicine, Wonju, Korea (the Republic of), 3 College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, Korea (the Republic of) and 4 Wellskin Research Center, Dankook University Hospital, Cheonan, Korea (the Republic of) The proportions of ceramides with very long-chain fatty acids (FAs) has been known to be reduced in the stratum corneum of patients with atopic dermatitis and psoriasis. It could negatively affects the proper function of skin barrier as well. Meanwhile, it has been reported that photoaged murine skin displayed abnormal barrier function and decreased lipid levels of stratum corneum. So, to further know the alteration of FA chain length of ceramides and lipid synthesis-related enzymes after long-term UV exposure, the profile of ceramide chain length was analyzed in photoaged mouse epidermis. Compared to age-matched untreated skin, some of unbound ceramides with longer than 26 carbons (such as ceramide NP and NDS) were significantly decreased, but bound ceramides (such as ceramide OS) only showed a decreasing tendency. There was a significantly decrease in mRNA expression of FA elongase ELOVL1 and ceramide synthase 3 in the photoaged murine epidermis. These enzymes are essentially involved in the formation of long-chain ceramides. To investigate the in vitro effect of low dose repeated UV exposure, the expression of lipid synthetic enzymes and their known transcription factors (such as PPARs and LXRa) was assessed after 6 times of low dose (5mJ/ cm2) of UVB radiation exposures to human epidermal keratinocyte. The expression of LXRa and PPARg was significantly suppressed in this in vitro photoaging model. Our results demonstrate ceramide chain length may be negatively affected in photoaged skin, as like atopic dermatitis.
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