1,25-(OH 2 )D 3 Alters the Transforming Growth Factor  Signaling Pathway in Renal Tissue J. K. Aschenbrenner, M.D.,* H. W. Sollinger, M.D., Ph.D.,* B. N. Becker, M.D.,† and D. A. Hullett, Ph.D.* ,1 *Departments of Surgery and †Medicine, University of Wisconsin, Madison, Wisconsin 53792 Submitted for publication November 10, 2000; published online August 21, 2001 Background. 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) plays an important role in regulating immune re- sponses, in addition to its effects on bone metabolism. The cytokine transforming growth factor  (TGF-) reg- ulates diverse biological processes, including cellular proliferation and differentiation, immune modulation, and modulation of extracellular matrix deposition. 1,25- (OH) 2 D 3 interacts in vitro with Smad proteins, impor- tant regulators of TGF- signal transduction. We hy- pothesized that exogenous 1,25-(OH) 2 D 3 would alter levels of TGF- 1 and TGF- 1 signaling proteins in renal tissue. Methods. C57BL6 mice and Lewis rats were placed on diets with or without 1,25-(OH) 2 D 3 for 14 days. Re- nal lysates were examined for TGF- 1 , vitamin D re- ceptor (VDR), and Smad3 protein levels using a cell proliferation assay and Western blot analysis. Coim- munoprecipitation was used to determine if any inter- action between VDR and Smad3 proteins occurs in vivo. Reverse transcription–polymerase chain reac- tion (RT-PCR) was used to assess messenger RNA (mRNA) levels for all of these molecules. Results. Vitamin D supplementation decreased VDR and Smad3 protein levels. Coimmunoprecipitation of VDR and Smad3 revealed a Smad3–VDR interaction in vivo. Vitamin D–treated rats had a significant (P  0.001) reduction in bioactive renal TGF- 1 . RT-PCR demonstrated no difference in mRNA expression for either VDR or TGF- 1 . Conclusion. Our results suggest that vitamin D has a significant effect in regulating levels of bioactive TGF- 1 and appears to affect aspects of the TGF- 1 signaling system. These effects, in combination with the immunomodulatory actions of vitamin D, may al- ter the evolution of chronic rejection in renal trans- plants. © 2001 Academic Press Key Words: vitamin D; 1,25-(OH) 2 D 3 ; renal transplan- tation; rejection; chronic allograft nephropathy; TGF-  1 ; Smad3; smad proteins; vitamin D receptor; immunosuppression. INTRODUCTION 1,25-Dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the active form of vitamin D, plays an important immunomodu- latory role in addition to its classic effects on bone and calcium metabolism [1]. 1,25-(OH) 2 D 3 affects the devel- opment and progression of autoimmune disease [2] and inhibits T-cell proliferation [3]. It also affects cytotoxic T-lymphocyte development, cytokine expression in vitro, and dendritic cell activation and function [4]. Finally, 1,25-(OH) 2 D 3 decreases major histocompetibil- ity complex class II molecule expression [5] and sup- presses both T helper cell type 1 and 2 cytokine pro- duction [6]. Transforming growth factor -1 (TGF- 1 ) is a com- plex cytokine that regulates diverse biological pro- cesses, including cellular proliferation [7] and differen- tiation [8], immune modulation [5, 9], and extracellular matrix remodeling [10, 11]. Chronic allograft nephrop- athy (CAN) is a complex disease entity resulting from interactions between humoral and cellular immune re- sponses and nonimmunologic injury. These all lead ultimately to intragraft fibrosis and a decline in renal function. TGF- 1 plays a significant role in CAN [12]. TGF- 1 has a complex but ubiquitous signaling sys- tem that relies on Smad proteins as downstream reg- ulators in the signal transduction pathway. These pro- teins are classified according to their known functional characteristics [8]. Specifically, the regulatory Smad proteins Smad2 and Smad3 form a complex with the 1 To whom correspondence and reprint requests should be ad- dressed at 600 Highland Avenue CSC H4/3, Madison, WI 53792. Fax: 608-265-9144. Email: hullett@surgery.wisc.edu. Journal of Surgical Research 100, 171–175 (2001) doi:10.1006/jsre.2001.6221, available online at http://www.idealibrary.com on 171 0022-4804/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.