Gene Expression Analysis Reveals Novel Functions of Vitamin D and Glucocorticoids

Steroid hormones as Vitamin D and glucocorticoids regulate multiple biochemical pathways and physiological processes. The discovery of nuclear hormone receptors and their mode of action in recent years helped to elucidate the mechanism of steroid-mediated transcriptional control. Disturbances in the control of these complex networks of gene regulation can cause severe diseases. Since it allows monitoring changes in the expression of thousands of genes in parallel, microarray technology was the method of choice to study the influence of glucocorticoids and vitamin D on transcription more in detail. Establishing cDNA microarray technology was the first task in this project. Microarrays containing cDNA fragments probing for several hundred genes were tested by measuring changes in the transcription profile of cell lines after exposure to steroids. Treatment of human fetal osteoblasts (hfOB) with Cyr61, a factor recently found to be induced by vitamin D in bone tissue culture, activated genes that belong to the Notch and Hedgehog signalling pathways. These pathways were reported to be involved in bone growth and remodelling. The identification of target genes of Cyr61, previously associated with functions during bone fracture repair, shows new signalling pathways controlled by vitamin D and Cyr61 in bone. The development and production of high-density cDNA arrays with nearly 21.000 probes allowed genome-wide transcriptional profiling in mouse. These microarrays were used to analyse the effect of glucocorticoid treatment of insulin-secreting beta cells of pancreatic islets. Corticosterone increased the secretion of insulin and induced genes known as markers for stress conditions, inflammation and a pre-diabetic state. The main project of this thesis was the analysis of transcriptional regulation by vitamin D in vivo. Tissues, especially kidney and heart from wild type (WT) and transgenic mice expressing a transcriptionally inactive vitamin D receptor (VDRKO mice) were analysed for changes in their gene expression profile. Under normal dietary conditions, VDRKO mice develop a phenotype that suffers from severe rickets, hypocalcemia, hyperparathyroidism, alopecia, impaired growth and bone formation. The role of vitamin D action in lipid metabolism and in the development of metabolic diseases like adiposity, hypertension or diabetes were clearly reflected in the functional annotations of differentially expressed genes in kidneys from VDRKO mice. Lipogenic genes were significantly up-regulated, apparently through a calcitriol-induced increase of intracellular calcium via VDR-independent mechanisms. Simultaneously, a loss of repression through the vitamin D receptor enhances the expression of uncoupling protein 1 (UCP1). Mitochondrial uncoupling increases the dissipation of energy. A resulting lower efficiency of ATP production can impair insulin signalling. The previously reported activation of the renin-angiotensin system in VDRKO mice was confirmed. In addition, this study identified the induction of a number of marker genes for hypertension and related diseases. The heart, a non-classical target organ of vitamin D, showed in the case of VDRKO mice changes in gene expression that correspond to the observations in kidney, the key organ of the vitamin D endocrine system. As in kidney, expression of lipogenic genes was significantly increased, together with markers for inflammation, stress response, cardiac hypertrophy and myopathy. Genes associated with the development of hypertrophy and metabolic switching from lipids to glucose and glutamin as energy carriers were activated. Strong induction of calgranulins and vitamin D-binding protein (DBP) show the in vivo impact of vitamin D on the immune response in heart for the first time. All studies in this thesis contribute to the development of microarray technology and its application to investigate the role of steroids in the development of multifactorial diseases. It gives evidence that impairment of the vitamin D endocrine system can affect the regulation of factors controlling central metabolic functions. Novel target genes and pathways regulated by vitamin D through genomic and non-genomic action were identified. The results from this study show that vitamin D is an important element connecting the so far unresolved association between rickets and hypertension, diabetes and cardiovascular diseases.