Knocking down raptor in human keratinocytes affects ornithine decarboxylase in a post-transcriptional Manner following ultraviolet B exposure
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Non-melanoma skin cancer (NMSC) is the most common form of cancer. Ultraviolet-B (UVB) radiation has been shown to be a complete carcinogen in the development of NMSC. The mammalian target of rapamycin complex 1 (mTORC1) is upregulated by UVB. Ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, is also upregulated in response to UVB. However, the interplay between these two pathways after UVB exposure remains unclear. The studies described here compare mRNA stability between normal human keratinocytes (HaCaT cells) and HaCaT cells with low levels of raptor to investigate whether the induction of ODC by UVB is dependent on mTORC1. We show that the knockdown of mTORC1 activity led to decreased levels of ODC protein both before and after exposure to 20 mJ/cm2 UVB. ODC mRNA was less stable in cells with decreased mTORC1 activity. Polysome profiles revealed that the initiation of ODC mRNA translation did not change in UVB-treated cells. We have shown that the ODC transcript is stabilized by the RNA-binding protein human antigen R (HuR). To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability in human keratinocytes exposed to UVB. We show an increased cytoplasmic localization of HuR after UVB exposure in wild-type cells. The ablation of HuR via CRISPR/Cas9 did not alter the stability of the ODC message, suggesting the involvement of other trans-acting factors. These data suggest that in human keratinocytes, ODC mRNA stability is regulated, in part, by an mTORC1-dependent mechanism after UVB exposure.
KeywordsOrnithine decarboxylase mTOR Post-transcriptional regulation mRNA stability
The authors would like to thank Dr. Scot Kimball (Penn State College of Medicine) for his help with the polysome profiles. Work in the authors’ laboratory was funded by grants from the National Institutes of Health (ES19242 to LMS, ES26471 to RPF) and funds from Penn State Berks to SLN.
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Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants performed by any of the authors. This article does not contain any studies with animals performed by any of the authors.
This article does not contain any studies with human participants performed by any of the authors.
- American Cancer Society (2016) Cancer facts and figures 2016Google Scholar
- Campistol JM, Eris J, Oberbauer R, Friend P, Hutchison B, Morales JM, Claesson K, Stallone G, Russ G, Rostaing L, Kreis H, Burke JT, Brault Y, Scarola JA, Neylan JF (2006) Sirolimus therapy after early cyclosporine withdrawal reduces the risk for cancer in adult renal transplantation. J Am Soc Nephrol 17(2):581–589. https://doi.org/10.1681/ASN.2005090993 CrossRefGoogle Scholar
- Chen SJ, Nakahara T, Takahara M, Kido M, Dugu L, Uchi H, Takeuchi S, Tu YT, Moroi Y, Furue M (2009) Activation of the mammalian target of rapamycin signalling pathway in epidermal tumours and its correlation with cyclin-dependent kinase 2. Br J Dermatol 160(2):442–445. https://doi.org/10.1111/j.1365-2133.2008.08903.x CrossRefGoogle Scholar
- Fernau NS, Fugmann D, Leyendecker M, Reimann K, Grether-Beck S, Galban S, Ale-Agha N, Krutmann J, Klotz LO (2010) Role of HuR and p38MAPK in ultraviolet B-induced post-transcriptional regulation of COX-2 expression in the human keratinocyte cell line HaCaT. J Biol Chem 285(6):3896–3904. https://doi.org/10.1074/jbc.M109.081430 CrossRefGoogle Scholar
- Guan BJ, Krokowski D, Majumder M, Schmotzer CL, Kimball SR, Merrick WC, Koromilas AE, Hatzoglou M (2014) Translational control during endoplasmic reticulum stress beyond phosphorylation of the translation initiation factor eIF2a. J Biol Chem 289(18):12593–12611. https://doi.org/10.1074/jbc.M113.543215 CrossRefGoogle Scholar
- Huang C, Li J, Ke Q, Leonard SS, Jiang BH, Zhong XS, Costa M, Castranova V, Shi X (2002) Ultraviolet-induced phosphorylation of p70(S6K) at Thr(389) and Thr(421)/Ser(424) involves hydrogen peroxide and mammalian target of rapamycin but not Akt and atypical protein kinase C. Cancer Res 62(20):5689–5697Google Scholar
- Nowotarski SL, Feehan RP, Presloid C, Shantz LM (2018) Knockout of Raptor destabilizes ornithine decarboxylase mRNA and decreases binding of HuR to the ODC transcript in cells exposed to ultraviolet-B irradiation. Biochem Biophys Res Commun 505(4):1022–1026. https://doi.org/10.1016/j.bbrc.2018.10.019 CrossRefGoogle Scholar
- O’Brien TG (1976) The induction of ornithine decarboxylase as an early, possibly obligatory, event in mouse skin carcinogenesis. Cancer Res 36(7 PT 2):2644–2653Google Scholar
- O’Brien TG, Megosh LC, Gilliard G, Soler AP (1997) Ornithine decarboxylase overexpression is a sufficient condition for tumor promotion in mouse skin. Cancer Res 57(13):2630–2637Google Scholar
- Origanti S, Shantz LM (2007) Ras transformation of RIE-1 cells activates cap-independent translation of ornithine decarboxylase: regulation by the Raf/MEK/ERK and phosphatidylinositol 3-kinase pathways. Cancer Res 67(10):4834–4842. https://doi.org/10.1158/0008-5472.CAN-06-4627 CrossRefGoogle Scholar
- Patursky-Polischuk I, Stolovich-Rain M, Hausner-Hanochi M, Kasir J, Cybulski N, Avruch J, Ruegg MA, Hall MN, Meyuhas O (2009) The TSC-mTOR pathway mediates translational activation of TOP mRNAs by insulin largely in a raptor- or rictor-independent manner. Mol Cell Biol 29(3):640–649. https://doi.org/10.1128/MCB.00980-08 CrossRefGoogle Scholar
- Rosen CF, Gajic D, Drucker DJ (1990a) Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes. Cancer Res 50(9):2631–2635Google Scholar
- Zabala-Letona A, Arruabarrena-Aristorena A, Martin-Martin N, Fernandez-Ruiz S, Sutherland JD, Clasquin M, Tomas-Cortazar J, Jimenez J, Torres I, Quang P, Ximenez-Embun P, Bago R, Ugalde-Olano A, Loizaga-Iriarte A, Lacasa-Viscasillas I, Unda M, Torrano V, Cabrera D, van Liempd SM, Cendon Y, Castro E, Murray S, Revandkar A, Alimonti A, Zhang Y, Barnett A, Lein G, Pirman D, Cortazar AR, Arreal L, Prudkin L, Astobiza I, Valcarcel-Jimenez L, Zuniga-Garcia P, Fernandez-Dominguez I, Piva M, Caro-Maldonado A, Sanchez-Mosquera P, Castillo-Martin M, Serra V, Beraza N, Gentilella A, Thomas G, Azkargorta M, Elortza F, Farras R, Olmos D, Efeyan A, Anguita J, Munoz J, Falcon-Perez JM, Barrio R, Macarulla T, Mato JM, Martinez-Chantar ML, Cordon-Cardo C, Aransay AM, Marks K, Baselga J, Tabernero J, Nuciforo P, Manning BD, Marjon K, Carracedo A (2017) mTORC1-dependent AMD1 regulation sustains polyamine metabolism in prostate cancer. Nature 547(7661):109–113. https://doi.org/10.1038/nature22964 CrossRefGoogle Scholar