Lineage specific effects of glucocorticoids in mesenchymal stem cell differentiation

Multipotent mesenchymal stem cells (MSCs) in the bone marrow play a central role in maintaining bone tissue homeostasis by replenishing populations of skeletal tissue cells, including bone-forming osteoblasts and lipid-storing marrow adipocytes. Differentiation of MSCs is governed by distinct transcriptional networks which when activated by environmental stimuli drive expression of the lineage-selective gene programs required for osteoblast or marrow adipocyte maturation. Understanding how transcriptional networks respond to environmental stimuli may pave the road for therapeutic interventions into MSC differentiation with e.g. tissue regenerative purposes.

This thesis investigates how glucocorticoids through activation of the glucocorticoid receptor influence osteoblast and adipocyte differentiation. Glucocorticoids are steroid hormones naturally produced in the body which owing to their immunosuppressive properties are frequently used in the treatment of auto-immune and inflammatory diseases. While glucocorticoids are required for the differentiation of MSCs into osteoblasts and adipocytes, patients receiving glucocorticoid therapy suffer from skeletal deterioration and onset of osteoporosis as a consequence of impaired osteoblast differentiation (reviewed in Chapter 1).

This thesis employs a genomics approach to investigate how glucocorticoids influence osteoblast and adipocyte differentiation in a human telomerase-immortalized MSCs model system (hMSC-TERT4). Chapter 2 discusses technical aspects of chromatin immunoprecipitation sequencing (ChIP-seq), a central technique for global mapping of protein-DNA interactions. Chapter 3 investigates lineage-selective and dose-dependent effects of glucocorticoids on hMSC-TERT4 osteoblast and adipocyte differentiation. It demonstrates how activation of the glucocorticoid receptor during early 24 hour osteoblast and adipocyte differentiation is indispensable for establishment of early lineage-selective gene programs. In addition, it shows how high concentrations of glucocorticoids impair hMSC-TERT4 osteoblast differentiation by blunting activation of late-stage osteoblast differentiation genes. Chapter 4 investigates how the glucocorticoid receptor cross-talks with the vitamin D receptor, an additional activator of osteoblast differentiation, and reveals a generally cooperative relationship between receptors in hMSC-TERT4 cells following acute stimulation with receptor ligands. Chapter 5 investigates the molecular basis of glucocorticoid receptor mediated gene activation and repression. It employs a data-driven machine-learning approach developed in collaboration with the Babu group (Laboratory of Molecular Biology, UK) to identify characteristics of glucocorticoid receptor activated and repressed sites, and based on this proposes that activation and repression likely involve distinct mechanisms. Finally, Chapter 6 presents results from a collaboration with the Kalkhoven group (University Medical Center Utrecht, NL) in which we have characterized how two novel familial partial lipodystrophy type 3 causing mutations in the peroxisome proliferator activated receptor . (PPAR.) affect this receptors ability to regulate target gene expression. Using ChIP- and RNA-seq we could show that the two mutations in a similar manner partially impair the ability of PPAR. to bind to PPAR response elements in the DNA and in turn activate target genes.