Protective effects of novel derivatives of vitamin D and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms
Authors: Tae-Kang Kim
Zorica Janjetovic
Uraiwan Panich
Robert C Tuckey
Wei Li
Edith K Y Tang
Michael F Holick
John A D&#x27;Orazio
Stuart G Jarrett
Anyamanee Chaiprasongsuk
Andrzej T Slominski
Journal: Redox Biology (2019)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: Epidermal keratinocytes; Lumisterol (L(3)); Nuclear factor E2-related factor 2 (Nrf2); Photoprotective effects; Ultraviolet B (UVB)
Affiliations: Department of Dermatology, University of Alabama at Birmingham, USA; Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand., Department of Dermatology, University of Alabama at Birmingham, USA., Department of Toxicology and Cancer Biology, The Markey Cancer Center, College of Medicine, University of Kentucky, Lexington, KY, USA., Department of Medicine, Boston University, MA, USA., School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia., Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand., Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN, USA., Department of Dermatology, University of Alabama at Birmingham, USA; VA Medical Center, Birmingham, AL, USA. Electronic address: .

Abstract:  We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents. 

Retracted: No

PubPeer comments:
 Could the authors please comment on the apparent splices in Figure 10B, although a splice seems to appear in a similar location on each band, they do not appear to be aligned. *** ![file] , 
 https: retractionwatch.com 2020 08 28 university-of-kentucky-demotes-cancer-researcher-following-finding-of-misconduct-by-scientist-in-his-lab , 
 #1 These are there two blots run simultaneously and then combined. It is in the same position. Blots are not stretched proportionately, so it appears it is in different position. If you found number of bands before and after, it is the same. The proteins were from the same experiment and separated simultaneously on two gels, because there was not enough space on the same gel. They were run simultaneously, and than submitted for WB and incubation with antibodies, simultaneously. , 
 #1 These are there two blots run simultaneously and then combined. It is in the same position. Blots are not stretched proportionately, so it appears it is in different position. If you found number of bands before and after, it is the same. The proteins were from the same experiment and separated simultaneously on two gels, because there was not enough space on the same gel. They were run simultaneously, and than submitted for WB and incubation with antibodies, simultaneously. Should we align them, or the explanation is sufficient? , 
 #2 These are there two blots run simultaneously and then combined. It is in the same position. Blots are not stretched proportionately, so it appears it is in different position. If you found number of bands before and after, it is the same. The proteins were from the same experiment and separated simultaneously on two gels, because there was not enough space on the same gel. They were run simultaneously, and than submitted for WB and incubation with antibodies, simultaneously. Should we align them, or the explanation is sufficient , 
 #5 Thank you for your reply. I believe I understand your explanation. Do you have original uncropped scans? If yes, you may post those here or use a link to an external site (such as Imgur.com) for larger files. You may also wish to consult the journal about how to represent spliced images. Their current policy is below: **Image manipulations** u201cAll Western blots should be presented with molecular weights noted, replicated, quantified and with statistical analysis. Cropping of the image is acceptable but must be clearly indicated. Merging images together to give the appearance of one image is not acceptable. The method of normalization to total protein, or where appropriate, a loading control (e.g. cell signaling studies) should be explicitly stated in the text. Images may be subjected to analysis for manipulation prior to publication and authors may be requested to provide copies of the original data.u201d , 
 I think that it is a great idea. I already asked Dr Anyamani who is the first author and Dr Janjetovic to work on this. It , 
 ![file] ![file] The jpg files are uploaded, I believe. Can you confirm. Thank you for allowing to deposit raw data , 
 #8 Thank you for sharing. Could you post a high resolution original image of the right hand Lamin A band without the red box? ![file] , 
 I will ask to prepare such image. You have 1 MB limit to deposit, which I learned hard way when you system was not allowing for uploading. But the image of proper size will be prepared , 
 #10 Thank you. You should use a link to an external site (such as Imgur.com) for larger files, and Imgur is free. File size limits: u201cNon-animated images over 1MB for anonymous uploads and 5MB for account holders will be lossily compressed. All non-animated images receive lossless compression that reduces filesize while maintaining quality.u201c , 
 ![file] The file in proper format is uploaded. Can you double-check , 
 #12 Dr. Slominski: The image you provided appears to show a splice, evidenced by the sharp change in contrast in the perimeter around the band. I recommend reviewing the original scans carefully to rule out possible deliberate manipulation. *** Inverse ![file] *** Original ![file] , 
 #13 I believe the line should be from the membrane line. You should know x-ray film is put on PVDF membrane which is is cut using scissors. Very thin height difference make the line when you develop, What do you think? Hopefully, you understand my explanation. , 
 I believe that the entire membrane was placed on x-ray film, and different exposures were done, which was standard in our lab. Yes, we still used traditional exposure to X-ray with film development. More work, but good for keeping documentation that also eliminated a possibility of electronic enhancement or inhibition. You just have few X-ray films developed and the best exposure is chosen for photographing. However, recently we started to use digitalized counter. Faster and more convenient. I do not believe that membrane was cut with scissors since they are re-probed with different antibodies after striping. However, on membranes we were placing srarap to keep membranes wet and not to destroy the film. I can check with the first author, but she is in different institution on different continent and may not remember, after 3 years. , 
 As explained by Professor, I would like to add further comments. The PVDF blot was visualized by the X-ray film. The edge on the X-ray film should be the PVDF blot which was cut prior to expose with the X-ray film. We used ECL reagent for the western blot detection of protein bound to HRP conjugated antibodies in the lab. Since the ECL fluorescence staining was rapidly faded in my blot, I quickly inserted the blot into the film case. That would make the edged appearance of PVDF blot and shifted scan in some area on the X-ray film. My sincere apologies for this cause the images confuse the readers. ,