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Scotin: A new p63 target gene expressed during epidermal differentiation.

Zocchi L, Bourdon JC, Codispoti A, Knight R, Lane DP, Melino G, Terrinoni A - Biochem. Biophys. Res. Commun. (2007)

Bottom Line: We detected Scotin upregulation in primary keratinocyte cell lines committed to differentiate.In this paper we also show that Scotin is expressed in the supra basal layer of the epidermis in parallel with TAp63, but not DeltaNp63 expression.We conclude that Scotin is a new p63 target gene induced during epithelial differentiation, a complex process that also involves ER stress induction.

View Article: PubMed Central - PubMed

Affiliation: IDI-IRCCS Biochemistry Laboratory, c/o University of Tor Vergata, Department of Experimental Medicine, Via Montpellier 1, 00133 Rome, Italy.

ABSTRACT
p63, a member of the p53 family, is transcribed from two different promoters giving rise to two different proteins: TAp63 that contains the N-terminal transactivation domain and DeltaN that lacks this domain. In this article we describe a new target gene Scotin induced by TAp63 during epithelial differentiation. This gene was previously isolated as a p53-inducible proapoptotic gene and the protein is located in the endoplasmic reticulum and in the nuclear membrane. Scotin expression is induced in response to endoplasmic reticulum (ER) stress in a p53 dependent or independent manner. We detected Scotin upregulation in primary keratinocyte cell lines committed to differentiate. In this paper we also show that Scotin is expressed in the supra basal layer of the epidermis in parallel with TAp63, but not DeltaNp63 expression. We conclude that Scotin is a new p63 target gene induced during epithelial differentiation, a complex process that also involves ER stress induction.

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TAp63α induces Scotin expression (A) Saos-2 cells stably transfected with doxycycline-inducible vectors containing p53, TAp63α or ΔNp63α were induced with doxycycline for 6 and 12 h. The over-expression of the selected p53 family members were confirmed by Western blot. RTqPCR shows that p53 and TAp63α expression, not ΔNp63α, triggers Scotin induction. (B) Saos-2 cells were transfected (12 and 24 h) with a TAp63 expression vector and were then immunostained for p63 (green) and Scotin (red). In TAp63 transfected cells, Scotin accumulates in the peri-nuclear area (possibly in the Golgi or endoplasmic reticulum). In contrast, p63−/− cells showed cytoplasmic Scotin staining. Bar = 20 μm.
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fig1: TAp63α induces Scotin expression (A) Saos-2 cells stably transfected with doxycycline-inducible vectors containing p53, TAp63α or ΔNp63α were induced with doxycycline for 6 and 12 h. The over-expression of the selected p53 family members were confirmed by Western blot. RTqPCR shows that p53 and TAp63α expression, not ΔNp63α, triggers Scotin induction. (B) Saos-2 cells were transfected (12 and 24 h) with a TAp63 expression vector and were then immunostained for p63 (green) and Scotin (red). In TAp63 transfected cells, Scotin accumulates in the peri-nuclear area (possibly in the Golgi or endoplasmic reticulum). In contrast, p63−/− cells showed cytoplasmic Scotin staining. Bar = 20 μm.

Mentions: Since p53 and TAp73α are able to induce Scotin expression and endoplasmic reticulum (ER) stress [18,20], we evaluated the presence of ER stress during the epidermal differentiation program in relation to the ability of p63 to activate Scotin expression. To investigate the ability of p63 to trigger Scotin expression, we performed RTqPCR experiments (Fig. 1A) using Tet-On cell lines which can be induced to express TAp63α, ΔNp63α or p53 (positive control) in the absence of interfering endogenous p53. Saos-2 cells are p53 and the expression of endogenous p63 is only weakly detectable with RT-PCR techniques. This allows complete exclusion of the contribution of p53 and minimizes the effect of endogenous p63 expression. The results confirmed the ability of p53 [18] and TAp63α to induce Scotin expression. ΔNp63α does not induce Scotin expression; rather ΔNp63α appears to reduce endogenous Scotin mRNA (Fig. 1A). This data has been confirmed by immunofluorescence on TAp63α transfected Saos-2 which shows Scotin expression only in cells where expression of TAp63 is clearly detectable (e.g. transfected; Fig. 1B). The induced Scotin appears as aggregates in the ER indicating the presence of ER stress, as previously shown [18,20]. These aggregates could be detected following TAp63α expression but not after the induction of ΔNp63α (data not shown).


Scotin: A new p63 target gene expressed during epidermal differentiation.

Zocchi L, Bourdon JC, Codispoti A, Knight R, Lane DP, Melino G, Terrinoni A - Biochem. Biophys. Res. Commun. (2007)

TAp63α induces Scotin expression (A) Saos-2 cells stably transfected with doxycycline-inducible vectors containing p53, TAp63α or ΔNp63α were induced with doxycycline for 6 and 12 h. The over-expression of the selected p53 family members were confirmed by Western blot. RTqPCR shows that p53 and TAp63α expression, not ΔNp63α, triggers Scotin induction. (B) Saos-2 cells were transfected (12 and 24 h) with a TAp63 expression vector and were then immunostained for p63 (green) and Scotin (red). In TAp63 transfected cells, Scotin accumulates in the peri-nuclear area (possibly in the Golgi or endoplasmic reticulum). In contrast, p63−/− cells showed cytoplasmic Scotin staining. Bar = 20 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2691585&req=5

fig1: TAp63α induces Scotin expression (A) Saos-2 cells stably transfected with doxycycline-inducible vectors containing p53, TAp63α or ΔNp63α were induced with doxycycline for 6 and 12 h. The over-expression of the selected p53 family members were confirmed by Western blot. RTqPCR shows that p53 and TAp63α expression, not ΔNp63α, triggers Scotin induction. (B) Saos-2 cells were transfected (12 and 24 h) with a TAp63 expression vector and were then immunostained for p63 (green) and Scotin (red). In TAp63 transfected cells, Scotin accumulates in the peri-nuclear area (possibly in the Golgi or endoplasmic reticulum). In contrast, p63−/− cells showed cytoplasmic Scotin staining. Bar = 20 μm.
Mentions: Since p53 and TAp73α are able to induce Scotin expression and endoplasmic reticulum (ER) stress [18,20], we evaluated the presence of ER stress during the epidermal differentiation program in relation to the ability of p63 to activate Scotin expression. To investigate the ability of p63 to trigger Scotin expression, we performed RTqPCR experiments (Fig. 1A) using Tet-On cell lines which can be induced to express TAp63α, ΔNp63α or p53 (positive control) in the absence of interfering endogenous p53. Saos-2 cells are p53 and the expression of endogenous p63 is only weakly detectable with RT-PCR techniques. This allows complete exclusion of the contribution of p53 and minimizes the effect of endogenous p63 expression. The results confirmed the ability of p53 [18] and TAp63α to induce Scotin expression. ΔNp63α does not induce Scotin expression; rather ΔNp63α appears to reduce endogenous Scotin mRNA (Fig. 1A). This data has been confirmed by immunofluorescence on TAp63α transfected Saos-2 which shows Scotin expression only in cells where expression of TAp63 is clearly detectable (e.g. transfected; Fig. 1B). The induced Scotin appears as aggregates in the ER indicating the presence of ER stress, as previously shown [18,20]. These aggregates could be detected following TAp63α expression but not after the induction of ΔNp63α (data not shown).

Bottom Line: We detected Scotin upregulation in primary keratinocyte cell lines committed to differentiate.In this paper we also show that Scotin is expressed in the supra basal layer of the epidermis in parallel with TAp63, but not DeltaNp63 expression.We conclude that Scotin is a new p63 target gene induced during epithelial differentiation, a complex process that also involves ER stress induction.

View Article: PubMed Central - PubMed

Affiliation: IDI-IRCCS Biochemistry Laboratory, c/o University of Tor Vergata, Department of Experimental Medicine, Via Montpellier 1, 00133 Rome, Italy.

ABSTRACT
p63, a member of the p53 family, is transcribed from two different promoters giving rise to two different proteins: TAp63 that contains the N-terminal transactivation domain and DeltaN that lacks this domain. In this article we describe a new target gene Scotin induced by TAp63 during epithelial differentiation. This gene was previously isolated as a p53-inducible proapoptotic gene and the protein is located in the endoplasmic reticulum and in the nuclear membrane. Scotin expression is induced in response to endoplasmic reticulum (ER) stress in a p53 dependent or independent manner. We detected Scotin upregulation in primary keratinocyte cell lines committed to differentiate. In this paper we also show that Scotin is expressed in the supra basal layer of the epidermis in parallel with TAp63, but not DeltaNp63 expression. We conclude that Scotin is a new p63 target gene induced during epithelial differentiation, a complex process that also involves ER stress induction.

Show MeSH