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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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LincRNA-p21 is regulated at the transcriptional level and is p53-dependent in mouse and human keratinocytes and mouse skin in vivo. (a) Balb/MK2 keratinocytes were treated with actinomycin D, exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (b) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and p53 protein levels were measured by immunoblot analysis. (c) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (d) K5Cre+/tg and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and p53 protein levels were measured by immunoblot analysis. (e) K5Cre+/tg and K5Cre+/tg;p53flox/flox mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and lincRNA-p21 transcripts measured. (f) MT2.5, MT2.6 and Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and collected 8 h later. (g) NHEK and HaCaT cells were exposed to 10 mJ/cm2 UVB and collected 12 h later. LincRNA-p21 transcript levels were determined by TaqMan real-time PCR. Expression of lincRNA-p21 was normalized to β-actin. Data expressed in a, c, e, f and g as the mean ±S.D. N ≥3, *P<0.05 significantly different compared to UVB exposed control as determined by the student t-test
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fig2: LincRNA-p21 is regulated at the transcriptional level and is p53-dependent in mouse and human keratinocytes and mouse skin in vivo. (a) Balb/MK2 keratinocytes were treated with actinomycin D, exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (b) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and p53 protein levels were measured by immunoblot analysis. (c) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (d) K5Cre+/tg and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and p53 protein levels were measured by immunoblot analysis. (e) K5Cre+/tg and K5Cre+/tg;p53flox/flox mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and lincRNA-p21 transcripts measured. (f) MT2.5, MT2.6 and Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and collected 8 h later. (g) NHEK and HaCaT cells were exposed to 10 mJ/cm2 UVB and collected 12 h later. LincRNA-p21 transcript levels were determined by TaqMan real-time PCR. Expression of lincRNA-p21 was normalized to β-actin. Data expressed in a, c, e, f and g as the mean ±S.D. N ≥3, *P<0.05 significantly different compared to UVB exposed control as determined by the student t-test

Mentions: To determine whether lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment, we treated Balb/MK2 keratinocytes with actinomycin D, an inhibitor of transcription, and then exposed the cells to UVB and collected the cells 12 h later. As shown in Figure 2a, actinomycin D blocked the increase in UVB-induced lincRNA-p21 transcripts indicating that lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment. Next, we examined the role of p53 on the regulation of UVB-induced lincRNA-p21 expression in Balb/MK2 keratinocytes in culture. UVB-treated p53 knockdown keratinocytes (Figure 2b) were significantly impaired in their ability to induce lincRNA-p21 transcripts (Figure 2c). Next, we examined the role of p53 on the regulation of UVB-induced lincRNA-p21 expression in vivo in mouse epidermis using K5Cre+/tg;p53flox/flox mice. In this mouse model, the keratin 5 (K5) promoter directs Cre recombinase expression to the epidermis.34 UVB-treated K5Cre+/tg;p53flox/flox mice SKH-1 mice lacking p53 in their epidermis (Figure 2d) displayed significantly decreased levels of epidermal lincRNA-p21 transcripts compared with UVB-treated K5Cre+/tg mice (Figure 2e). Although the majority (~85%) of UVB-induced lincRNA-p21 in mouse skin in vivo occurs through a p53-dependent pathway, there also appears to be a minor (~15%) p53-independent pathway involved in the UVB regulation of lincRNA-p21 transcript levels (Figures 2e). We also observed that mouse skin SCC lines that are defective in p53 signaling (MT2.5 and MT2.6)35 were unable to induce lincRNA-p21 in response to UVB radiation compared with mouse keratinocytes (Figure 2f). Likewise, HaCaT cells which are a spontaneously immortalized human keratinocyte cell line that contain two alleles of mutant p5336 also displayed significantly impaired UVB induction of lincRNA-p21 when compared with similarly treated NHEKs (Figure 2g). Collectively, these data demonstrate that lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment and that the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is dependent upon p53.


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)

LincRNA-p21 is regulated at the transcriptional level and is p53-dependent in mouse and human keratinocytes and mouse skin in vivo. (a) Balb/MK2 keratinocytes were treated with actinomycin D, exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (b) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and p53 protein levels were measured by immunoblot analysis. (c) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (d) K5Cre+/tg and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and p53 protein levels were measured by immunoblot analysis. (e) K5Cre+/tg and K5Cre+/tg;p53flox/flox mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and lincRNA-p21 transcripts measured. (f) MT2.5, MT2.6 and Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and collected 8 h later. (g) NHEK and HaCaT cells were exposed to 10 mJ/cm2 UVB and collected 12 h later. LincRNA-p21 transcript levels were determined by TaqMan real-time PCR. Expression of lincRNA-p21 was normalized to β-actin. Data expressed in a, c, e, f and g as the mean ±S.D. N ≥3, *P<0.05 significantly different compared to UVB exposed control as determined by the student t-test
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fig2: LincRNA-p21 is regulated at the transcriptional level and is p53-dependent in mouse and human keratinocytes and mouse skin in vivo. (a) Balb/MK2 keratinocytes were treated with actinomycin D, exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (b) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and p53 protein levels were measured by immunoblot analysis. (c) Balb/MK2 cells were transfected with siRNA to p53 or control siRNA, and 48 h post transfection, cells were exposed to 10 mJ/cm2 UVB, collected 12 h later and lincRNA-p21 transcripts measured. (d) K5Cre+/tg and K5Cre+/tg;p53flox/flox SKH-1 mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and p53 protein levels were measured by immunoblot analysis. (e) K5Cre+/tg and K5Cre+/tg;p53flox/flox mice were treated with 200 mJ/cm2 UVB and epidermis collected 9 h later and lincRNA-p21 transcripts measured. (f) MT2.5, MT2.6 and Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and collected 8 h later. (g) NHEK and HaCaT cells were exposed to 10 mJ/cm2 UVB and collected 12 h later. LincRNA-p21 transcript levels were determined by TaqMan real-time PCR. Expression of lincRNA-p21 was normalized to β-actin. Data expressed in a, c, e, f and g as the mean ±S.D. N ≥3, *P<0.05 significantly different compared to UVB exposed control as determined by the student t-test
Mentions: To determine whether lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment, we treated Balb/MK2 keratinocytes with actinomycin D, an inhibitor of transcription, and then exposed the cells to UVB and collected the cells 12 h later. As shown in Figure 2a, actinomycin D blocked the increase in UVB-induced lincRNA-p21 transcripts indicating that lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment. Next, we examined the role of p53 on the regulation of UVB-induced lincRNA-p21 expression in Balb/MK2 keratinocytes in culture. UVB-treated p53 knockdown keratinocytes (Figure 2b) were significantly impaired in their ability to induce lincRNA-p21 transcripts (Figure 2c). Next, we examined the role of p53 on the regulation of UVB-induced lincRNA-p21 expression in vivo in mouse epidermis using K5Cre+/tg;p53flox/flox mice. In this mouse model, the keratin 5 (K5) promoter directs Cre recombinase expression to the epidermis.34 UVB-treated K5Cre+/tg;p53flox/flox mice SKH-1 mice lacking p53 in their epidermis (Figure 2d) displayed significantly decreased levels of epidermal lincRNA-p21 transcripts compared with UVB-treated K5Cre+/tg mice (Figure 2e). Although the majority (~85%) of UVB-induced lincRNA-p21 in mouse skin in vivo occurs through a p53-dependent pathway, there also appears to be a minor (~15%) p53-independent pathway involved in the UVB regulation of lincRNA-p21 transcript levels (Figures 2e). We also observed that mouse skin SCC lines that are defective in p53 signaling (MT2.5 and MT2.6)35 were unable to induce lincRNA-p21 in response to UVB radiation compared with mouse keratinocytes (Figure 2f). Likewise, HaCaT cells which are a spontaneously immortalized human keratinocyte cell line that contain two alleles of mutant p5336 also displayed significantly impaired UVB induction of lincRNA-p21 when compared with similarly treated NHEKs (Figure 2g). Collectively, these data demonstrate that lincRNA-p21 is regulated at the transcriptional level in response to UVB treatment and that the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is dependent upon p53.

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