Molecular Cloning and Characterization of Bovine CYP26A1 Promoter

국문 초록 (Abstract)

레티노산(RA)는 많은 유형의 세포에서 성장 및 발달에 중요한 역할을 수행하며 생체 활성화에 적합한 RA의 농도는 CYP26A1 등 여러 가지 효소에 의해 조절된다. CYP26A1의 발현은 RA에 의해서 조절되며 CYP26A1는 RA에 반응하는 유전자 중 하나이다. CYP26A1 유전자 클로닝은 여러 동물에서 보고되어 있지만, 소에서 CYP26A1 유전자의 클로닝은 아직 보고되지 않았다. 소로부터 CYP26A1의 프로모터 부위를 중합효소 연쇄반응을 이용하여 클로닝 한 후 다른 동물과 염기 서열 비교분석 결과 RARE DR-5 (TGAACTttgggTGAACT)의 존재를 확인하였고, DR-5의 염기서열은 분석한 종 에서 완전히 일치하였다. DR-5 motif를 함유한 소의 CYP26A1 프로모터 부위를luciferase리포터 유전자에 결합한 후 transient transfection에 의해 promoter 발현을 분석하였다. 폐 유래 세포주인 MTCC 세포에서 CYP26A1 promoter의 발현은 ATRA의 처리에 의하여 촉진되었다. CYP26A1 유전자의 발현은 ATRA 의존적으로 RAR-α 및 RAR-β에 의하여 현저하게 촉진되었다. 그러나 RAR-γ나 RXR-γ는 CYP26A1 발현에 별다른 영향을 미치지는 않았다. 또한 MTCC 세포주가 생산하는 내인성 CYP26A1 유전자 발현을 Q-RT-PCR로 분석한 결과 1-2일간의 ATRA 처리에 의해서는 현저한 영향을 받지 않으나, 3일 동안 ATRA를 처리한 샘플에서는 CYP26A1의 발현이 현저하게 감소하였다. 결론적으로, 소의 CYP26A1유전자의 프로모터 부위에 존재하는 DR-5 RARE는 RAR-α 및 RAR-β의 결합부위로 작용하여 MTCC 세포에서 CYP26A1 유전자 발현 조절과 RA signal의 조절에 관여하는 것을 확인하였다.

다국어 초록 (Multilingual Abstract)

The retinoic acid (RA) plays an important role in the growth and development of many cells, and bioactive RA concentration is regulated by several enzymes, including CYP26A1. The expression of the CYP26A1 gene is regulated by RA, and the CYP26A1 gene is one of the candidates for RA-responsive genes. Although CYP26A1 genes are cloned from several animals, cloning of the CYP26A1 gene from cows has not been reported yet. The promoter region of CYP26A1 from cows was cloned by PCR and analyzed by sequence alignment with human and mouse CYP26A1. The RA-responsive element (RARE), DR-5 (TGAACTttgggTGAACT), was located in this region and was perfectly conserved. The promoter region of bovine CYP26A1, which contains DR-5, was ligated to the luciferase reporter gene on transient transfection assays. The expression of CYP26A1-Luc promoter was activated by ATRA treatment in lung-derived mtCC cells. Co-transfection with RAR-α or -β with ATRA significantly activates the expression of CYP26A1-Luc promoter; however, it was less effective with either RAR-γ or RXR-γ. In addition, the endogenous gene expressions measured by Q-RT-PCR in mtCC cells were not significantly affected by ATRA treatment for 2 days; however, the expression of the endogenous CYP26A1 gene was diminished sharply at day 3 with ATRA treatment. In conclusion, the promoter region of bovine CYP26A1 contains conserved DR-5 RARE, which functions as a binding site for RAR-α or –β, and it is involved in the regulation of CYP26A1 gene expression and the control of RA signaling in mtCC cells.

목차 (Table of Contents)

• Introduction
• Materials and Methods
• Results and Discussion
• References
• 초록

참고문헌 (Reference)

1 Ray, M. K., "Transcriptional regulation of a mouse Clara cell-specific protein (mCC10) gene by the NKx transcription factor family member thyroid transcription factor 1 and cardiac muscle-specific homeobox protein (CSX)" 16 : 2056-2064, 1996

2 Loudig, O., "Transcriptional co-operativity between distant retinoic acid response elements in regulation of CYP26A1 inducibility" 392 : 241-248, 2005

3 Maden, M, "The role of retinoic acid in embryonic and post-embryonic development" 59 : 65-73, 2000

4 Thatcher, J. E., "The role of CYP26enzymes in retinoic acid clearance" 5 : 875-886, 2009

5 Abu-Abed, S., "The retinoic acid-metabolizing enzyme, CYP26A1, is essential for normal hindbrain patterning, vertebral identity, and development of posterior structures" 15 : 226-240, 2001

6 Van Heusden, J., "The antiproliferative activity of all-trans-retinoic acid catabolites and isomers is differentially modulated by liarozole-fumarate in MCF-7 human breast cancer cells" 77 : 1229-1235, 1998

7 Tang, X. H., "Retinoids, retinoic acid receptors, and cancer" 6 : 345-364, 2011

8 Freemantle, S. J., "Retinoids in cancer therapy and chemoprevention: promise meets resistance" 22 : 7305-7315, 2003

9 Bushue, N., "Retinoid pathway and cancer therapeutics" 62 : 1285-1298, 2010

10 Duester, G, "Retinoic acid synthesis and signaling during early organogenesis" 134 : 921-931, 2008

11 Arrieta, Ó., "Retinoic acid reduces chemotherapy-induced neuropathy in an animal model and patients with lung cancer" 77 : 987-995, 2011

12 Yamamoto, Y., "Regulation of CYP26 (cytochrome P450RAI) mRNA expression and retinoic acid metabolism by retinoids and dietary vitamin A in liver of mice and rats" 14 : 2119-2127, 2000

13 Chawla, A., "Nuclear receptors and lipid physiology: Opening the X-files" 294 : 1866-1870, 2001

14 Aranda, A., "Nuclear hormone receptors and gene expression" 81 : 1269-1304, 2001

15 Zhang, M., "Molecular characterization of a mouse short chain dehydrogenase/reductase active with all-trans-retinol in intact cells, mRDH1" 276 : 44083-44090, 2001

16 Klaassen, I., "Metabolism and growth inhibition of four retinoids in head and neck squamous normal and malignant cells" 85 : 630-635, 2001

17 Rohwedel, J., "Induction of cellular differentiation by retinoic acid in vitro" 165 : 190-202, 1999

18 Gomaa, M. S., "Homology model of human retinoic acid metabolising enzyme cytochrome P450 26A1 (CYP26A1): active site architecture and ligand binding" 21 : 361-369, 2007

19 Osanai, M., "Expression of the retinoic acid-metabolizing enzyme CYP26A1 limits programmed cell death" 67 : 1808-1817, 2005

20 Mongan, N. P., "Diverse actions of retinoid receptors in cancer prevention and treatment" 75 : 853-870, 2007

21 Glass, C. K, "Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers" 15 : 391-407, 1994

22 Ross, A. C., "Cytochrome P450s in the regulation of cellular retinoic acid metabolism" 31 : 65-87, 2011

23 Loudig, O., "Cytochrome P450RAI (CYP26)promoter: a distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism" 14 : 1483-1497, 2000

24 Orfanos, C. E., "Current use and future potential role of retinoids in dermatology" 53 : 358-388, 1997

25 Germain, P., "Co-regulator recruitment and the mechanism of retinoic acid receptor synergy" 415 : 187-192, 2002

26 Uehara, M., "CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse" 302 : 399-411, 2007

27 Ray, W. J., "CYP26, a novel mammalian cytochrome P450, is induced by retinoic acid and defines a new family" 272 : 18702-18708, 1997

28 Ozpolat, B., "All-trans-retinoic acid-induced expression and regulation of retinoic acid 4-hydroxylase (CYP26) in human promyelocytic leukemia" 70 : 39-47, 2002

29 Zhang, H., "All-trans retinoic acid (atRA) differentially induces apoptosis in matched primary and metastatic melanoma cells --a speculation on damage effect of atRA via mitochondrial dysfunction and cell cycle redistribution" 24 : 185-191, 2003

30 Rossetti, D., "A novel anti-ageing mechanism for retinol: induction of dermal elastin synthesis and elastin fibre formation" 33 : 62-69, 2011

31 Baes, M., "A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements" 14 : 1544-1552, 1994

32 Chambon, P, "A decade of molecular biology of retinoic acid receptors" 10 : 940-954, 1996

33 Aboulafia, D. M., "9-cis-retinoic acid capsules in the treatment of AIDS-related Kaposi sarcoma: results of a phase 2 multicenter clinical trial" 139 : 178-186, 2003

34 Maeng, S., "9-Cis-retinoic acid induces growth inhibition in retinoid-sensitive breast cancer and sea urchin embryonic cells via retinoid X receptor α and replication factor C3" 26 : 1821-1835, 2012