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Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes.

Hall JR, Messenger ZJ, Tam HW, Phillips SL, Recio L, Smart RC - Cell Death Dis (2015)

Bottom Line: Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes.We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death.We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA [2] Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, USA [3] Toxicology Program, North Carolina State University, Raleigh, NC, USA.

ABSTRACT
LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53(+/R172H) allele expressed in mouse epidermis (K5Cre(+/tg);LSLp53(+/R172H)) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

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Mutation of single p53 allele has a dominant-negative effect in vivo in mouse epidermis. (a) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermal lincRNA-p21 transcripts measured 9 h post UVB treatment. (b) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 100 mJ/cm2 UVB and the number of apoptotic interfollicular basal epidermal keratinocytes/cm skin were scored at 9 h post UVB treatment. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with UVB-treated K5Cre mice as determined by the student t-test
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fig5: Mutation of single p53 allele has a dominant-negative effect in vivo in mouse epidermis. (a) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermal lincRNA-p21 transcripts measured 9 h post UVB treatment. (b) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 100 mJ/cm2 UVB and the number of apoptotic interfollicular basal epidermal keratinocytes/cm skin were scored at 9 h post UVB treatment. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with UVB-treated K5Cre mice as determined by the student t-test

Mentions: Because the development of UVB-induced skin cancer often entails early oncogenic events involving the mutation of a single p53 allele and the escape from UVB-induced apoptosis, we examined whether a dominant inhibitory mutation in a single p53 allele (p53+/R172H) could greatly impair UVB-induced lincRNA-p21 transcript levels and apoptosis when compared with loss-of-function mutation involving deletion of a single p53 allele (p53+/−). Previous studies have shown that missense mutants of p53 in the heterozygous state can function as dominant-negative inhibitors of certain tumor-suppressive functions and retain normal wild-type p53 of others.37, 38 For example, in p53+/R172H cells, the mutant p53 does not have a dominant-negative effect on DNA damage-induced G1 arrest or p21 gene expression but does have a dominant-negative effect on gamma radiation-induced apoptosis.38 Therefore, we generated two genetically engineered mouse models (K5Cre+/tg;p53+/flox and K5Cre+/tg;LSLp53+/R172H)38, 39 to test whether the mutation of a single p53 allele (p53+/R172H) as opposed to the loss of a single p53 allele (p53+/−), both expressed under normal physiological control in vivo in epidermis of SKH-1 mice, will have a dominant-negative effect on UVB-induced lincRNA-p21 expression and keratinocyte apoptosis. In this mouse model, the keratin 5 (K5) promoter directs Cre recombinase expression to the epidermis.34 As shown in Figure 5a, UVB-induced lincRNA-p21 levels in K5Cre+/tg;LSLp53+/R172H mice were greatly decreased and were similar to the levels in K5Cre+/tg;p53flox/flox mice whereas lincRNA-p21 transcript levels in K5Cre+/tg and K5Cre+/tg;p53+/flox were similar. These results demonstrate a potent effect of the dominant inhibitory mutation of a p53 allele (p53+/R172H) on UVB-induced lincRNA-p21 transcript levels. Next, we examined whether the decreased levels of lincRNA-p21 transcripts in UVB-treated K5Cre+/tg;LSLp53+/R172H are associated with decreased p53-dependent apoptosis induced by UVB. As shown in Figure 5b, approximately 50% of UVB-induced apoptosis in mouse epidermis is dependent on p53. UVB-induced apoptosis was similarly decreased in K5Cre+/tg;p53flox/flox and K5Cre+/tg;LSLp53+/R172H mice indicating that the mutation in a single p53 allele (p53+/R172H) has a significant dominant-negative inhibitory effect on lincRNA-p21 transcription and apoptosis in vivo. These data further suggest that deregulation of lincRNA-p21 expression may be important in the early pro-oncogenic functions of p53 mutant keratinocytes during the development of skin cancer.


Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes.

Hall JR, Messenger ZJ, Tam HW, Phillips SL, Recio L, Smart RC - Cell Death Dis (2015)

Mutation of single p53 allele has a dominant-negative effect in vivo in mouse epidermis. (a) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermal lincRNA-p21 transcripts measured 9 h post UVB treatment. (b) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 100 mJ/cm2 UVB and the number of apoptotic interfollicular basal epidermal keratinocytes/cm skin were scored at 9 h post UVB treatment. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with UVB-treated K5Cre mice as determined by the student t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4385943&req=5

fig5: Mutation of single p53 allele has a dominant-negative effect in vivo in mouse epidermis. (a) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermal lincRNA-p21 transcripts measured 9 h post UVB treatment. (b) K5Cre+/tg, K5Cre+/tg;LSLp53+/R172H, K5Cre+/tg;p53+/flox and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 100 mJ/cm2 UVB and the number of apoptotic interfollicular basal epidermal keratinocytes/cm skin were scored at 9 h post UVB treatment. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with UVB-treated K5Cre mice as determined by the student t-test
Mentions: Because the development of UVB-induced skin cancer often entails early oncogenic events involving the mutation of a single p53 allele and the escape from UVB-induced apoptosis, we examined whether a dominant inhibitory mutation in a single p53 allele (p53+/R172H) could greatly impair UVB-induced lincRNA-p21 transcript levels and apoptosis when compared with loss-of-function mutation involving deletion of a single p53 allele (p53+/−). Previous studies have shown that missense mutants of p53 in the heterozygous state can function as dominant-negative inhibitors of certain tumor-suppressive functions and retain normal wild-type p53 of others.37, 38 For example, in p53+/R172H cells, the mutant p53 does not have a dominant-negative effect on DNA damage-induced G1 arrest or p21 gene expression but does have a dominant-negative effect on gamma radiation-induced apoptosis.38 Therefore, we generated two genetically engineered mouse models (K5Cre+/tg;p53+/flox and K5Cre+/tg;LSLp53+/R172H)38, 39 to test whether the mutation of a single p53 allele (p53+/R172H) as opposed to the loss of a single p53 allele (p53+/−), both expressed under normal physiological control in vivo in epidermis of SKH-1 mice, will have a dominant-negative effect on UVB-induced lincRNA-p21 expression and keratinocyte apoptosis. In this mouse model, the keratin 5 (K5) promoter directs Cre recombinase expression to the epidermis.34 As shown in Figure 5a, UVB-induced lincRNA-p21 levels in K5Cre+/tg;LSLp53+/R172H mice were greatly decreased and were similar to the levels in K5Cre+/tg;p53flox/flox mice whereas lincRNA-p21 transcript levels in K5Cre+/tg and K5Cre+/tg;p53+/flox were similar. These results demonstrate a potent effect of the dominant inhibitory mutation of a p53 allele (p53+/R172H) on UVB-induced lincRNA-p21 transcript levels. Next, we examined whether the decreased levels of lincRNA-p21 transcripts in UVB-treated K5Cre+/tg;LSLp53+/R172H are associated with decreased p53-dependent apoptosis induced by UVB. As shown in Figure 5b, approximately 50% of UVB-induced apoptosis in mouse epidermis is dependent on p53. UVB-induced apoptosis was similarly decreased in K5Cre+/tg;p53flox/flox and K5Cre+/tg;LSLp53+/R172H mice indicating that the mutation in a single p53 allele (p53+/R172H) has a significant dominant-negative inhibitory effect on lincRNA-p21 transcription and apoptosis in vivo. These data further suggest that deregulation of lincRNA-p21 expression may be important in the early pro-oncogenic functions of p53 mutant keratinocytes during the development of skin cancer.

Bottom Line: Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes.We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death.We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA [2] Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, USA [3] Toxicology Program, North Carolina State University, Raleigh, NC, USA.

ABSTRACT
LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53(+/R172H) allele expressed in mouse epidermis (K5Cre(+/tg);LSLp53(+/R172H)) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

Show MeSH
Related in: MedlinePlus