The Journal of Steroid Biochemistry and Molecular Biology
Vitamin D inhibition of pro-fibrotic effects of transforming growth factor β1 in lung fibroblasts and epithelial cells
Introduction
Dysregulated synthesis and tissue accumulation of matrix molecules are the hallmarks of fibrotic diseases and are directly responsible for organ destruction and abnormal physiology. In the lung, these changes may be seen in two anatomically distinct diseases: idiopathic pulmonary fibrosis (IPF) and post-transplant obliterative bronchiolitis (OB). Both IPF and OB carry poor prognoses, with mortality rates upwards of 50% at 3 years [1], [2], [3], and to date, there are no safe and effective treatments capable of halting and/or reversing the fibrotic processes in these diseases.
While not enough is known about the mechanisms responsible for these events, some hypotheses implicate an aberrant wound healing process in response to some known or unspecified injurious stimulus. Characteristically, there is fibroblast activation with a cellular phenotype typified by a resistance to apoptosis, the overproduction of connective tissue matrices, and the appearance of a contractile apparatus [4]. What triggers the induction of these putative myofibroblasts likely involves TGFβ, a pro-fibrotic cytokine consistently found in fibrotic tissues [5]. Interestingly, the changes in fibroblasts promoted by TGFβ also seem to be mimicked by epithelial cells under similar conditions [6]. Thus, a greater understanding of host and environmental factors influencing fibroproliferation and matrix expression in the lung is needed in order to identify novel targets for the development of effective therapies.
Vitamin D is a steroid pre-pro-hormone that requires sequential enzymatic modification, 25- and 1α-hydroxylation, in the liver and kidney, respectively, to have maximal biological activity as the hormone, 1,25(OH)2D. 1,25(OH)2D acts in an endocrine fashion on target organs (i.e. parathyroid, bone, intestine and kidney) to regulate bone, calcium, and phosphate metabolism; and its effects are mediated through a specific protein, the vitamin D receptor (VDR).
VDR is a ligand-dependent transcription factor belonging to the super-family of nuclear hormone receptors. VDR binds to its ligand, 1,25(OH)2D, dimerizes with the retinoid X receptor (RXR), and attaches to specific genomic sequences termed vitamin D response elements (VDREs). Like others in the nuclear hormone receptor class, VDRs are modular in structure, with an N-terminal transcriptional activation domain and C-terminal ligand-binding domain. The mid-region DNA binding domain, which consists of two zinc fingers, specifically recognizes DR3-type DNA motifs in target promoters to modulate gene expression. In point of fact, the expression of over two-hundred genes are controlled via 1,25(OH)2D/VDR-dependent pathways, either directly or indirectly, including genes that regulate proliferation, differentiation, and apoptosis, amongst others [7].
There does appear to be a role for 1,25(OH)2D in the “non-calcemic” homeostasis of extra-skeletal organs as suggested in a recent clinical review on vitamin D [7]. Evidence for this is provided by data demonstrating the expression of VDRs and 1α-hydroxylases in brain, prostate, breast, colon tissues, and circulating leukocytes. However, the exact role of vitamin D in such organs remains unelucidated and the biological consequences of vitamin D deficiency remain unclear. Of note, epidemiologic studies suggest a role for vitamin D deficiency in the pathogenesis of numerous chronic illnesses [7]. For one, there is circumstantial evidence that living at higher latitudes (where decreased solar UVB radiation is a cause of vitamin D deficiency) increases the risk of multiple sclerosis, Crohn's disease, hypertension and cardiovascular disease, hematological and solid organ malignancy, and asthma. The strongest data come from the cancer literature linking vitamin D deficiency with increased incidence of and mortality from colon, prostate, and breast cancer. Moreover, higher maternal intake of vitamin D during pregnancy was found in one study to be associated with a lower risk of recurrent wheeze in children at 3 years of age. Interestingly, a recent meta-analysis of 18 randomized clinical trials enrolling over 57,000 subjects suggested that intake of vitamin D supplements may decrease all-cause mortality [8].
Little is known about the role of vitamin D in lung or its impact on the function of lung cells. In particular, vitamin D status has not been systematically studied in patients with fibrotic lung disease. Unfortunately, such data are only found in studies of end-stage lung disease patients on a transplant waiting list containing a subgroup of pulmonary fibrotics [9], [10]. We speculate that vitamin D may influence lung fibrosis through direct effects on lung fibroblasts. Specifically, we hypothesize that vitamin D opposes intracellular pro-fibrogenic signals. To begin to address this hypothesis, we studied NIH3T3 fibroblastic cells and primary lung fibroblasts and found that they express functional vitamin D receptors. In these cells, vitamin D opposed the effects of transforming growth factor β1 (TGFβ1), a well-studied pro-fibrotic factor. Interestingly, vitamin D seems to have similar anti-TGFβ1 effects in lung epithelial cells thereby extending its protective functions to several pulmonary cells.
Section snippets
General reagents
The following antibodies were used against E-cadherin (BD Transduction Laboratories, San Jose, CA), α-smooth muscle actin (αSMA) (Sigma, St. Louis, MO), VDR (Affinity BioReagents, Golden, CO), procollagen type I (Novus Biologicals, Littleton, CO and Developmental Studies Hybridoma Bank, University of Iowa), fibronectin (Sigma), ZO-1 (Invitrogen, Carlsbad, CA), cytokeratin (DSHB, IA and Santa Cruz Biotechnology, Santa Cruz, CA), and PCNA (Cell Signal Technology, Danvers, MA). 1,25(OH)2D3 and
Lung fibroblasts express functional vitamin D receptors
To date, it remains unclear whether lung cells are capable of recognizing 1,25(OH)2D3 through the classic vitamin D receptor (VDR). To test this, we began by evaluating the expression of VDRs in lung tissue, and since fibroblasts are thought to be the predominant effector cell in organ fibrosis, we also assessed the expression of VDRs in this cell type. As depicted in Fig. 1, VDR mRNA and protein expression were detected in the NIH/3T3 cell line (confirming previous reports [20]), primary lung
Discussion
The role of vitamin D in lung remains largely unexplored. Specifically, it is unclear whether lung cells recognize vitamin D and whether vitamin D levels affect lung cell functions. We hypothesized that vitamin D influences pro-fibrogenic processes by targeting lung fibroblasts and tested this hypothesis in NIH/3T3 cells and primary murine lung fibroblasts. We demonstrated VDR expression in lung tissue homogenates and in lung fibroblasts. Furthermore, we found that the VDR localizes to the
Acknowledgements
This work was supported by NIH grant, 5K08HL080293 (C.W.), a research award from the Roche Organ Transplant Research Foundation (A.M.R.), and the American Heart Association (A.M.R.).
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