Edith Dalgaard posted an update 3 months, 1 week ago
We discovered that hMSC of patients suffering from severe osteoporosis display a disease-specific gene expression pattern that is distinct from the effects of organismic aging per se. Besides the induced expression of inhibitors of bone formation we detected promising new candidate genes for osteoporosis and even found evidence for reduced stem cell function. One of the main risk factors for developing primary osteoporosis is advanced age. Therefore, in the next step, we focused on gene expression patterns that were identical in hMSC-OP of elderly patients suffering from osteoporosis and hMSC-old of nonosteoporotic, elderly donors. As a new control group for microarray comparisons, we used hMSC of middle-aged donors. During aging, a continuous decrease in bone mass and bone density occurs and peaks in the development of primary osteoporosis in one of three women and one of eight men over the age of 50. Induced by a variety of risk factors like advanced age, loss of sex steroid production and unhealthy life style, recent research has largely unraveled the polygenetic nature and the multifaceted pathophysiology of this syndrome. Hitherto, approaches for studying the disease mostly consisted of whole genome association studies of BMD-associated gene loci as well as of manipulating expression of candidate genes in animal models or cells in vitro, followed by characterization of phenotypes. However, bone loss associated with increasing age is a continuous process not only caused by gene polymorphisms but very likely also by epigenetic modulations of gene expression changes that accompany aging. So far, analyses of these changes in primary cells of osteoporotic patients or in whole bone samples have been almost neglected. We analyzed the effect of primary osteoporosis on the source of bone regeneration and performed microarray hybridizations of hMSC of elderly patients suffering from severe osteoporosis and of donors of advanced age without any indication for the syndrome. We detected several genes connected to BMD with either reduced or increased expression in hMSC-OP including well-investigated susceptibility genes like LRP5, SPP1, COL1A1 and SOST. The latter codes for the osteocyte-specific protein Sclerostin, which acts as a WNT antagonist and is also controversially discussed as a BMP inhibitor. Upon release, the protein inhibits proliferation of MSC and osteoblasts, blocks osteogenic differentiation and even induces apoptosis in osteoblasts. Direct connections between the protein and osteoporosis have already been described: serum levels of Sclerostin were found enhanced in postmenopausal women and one of the upcoming treatments for osteoporosis is the application of anti- Sclerostin-antibodies. It is conceivable that the premature expression of SOST in osteoporotic stem cells auto-inhibits proliferation and self-renewal of hMSC-OP and thereby leads to the reduced ratios of formation to resorption observed in primary osteoporosis. Furthermore we also found higher expression of MAB21L2 in hMSC-OP in comparison to hMSC-old. QPCR revealed that, even though the expression was induced by advanced donor age itself, the transcription of MAB21L2 was even more triggered in osteoporotic stem cells. In Xenopus laevis gastrulae it was shown that MAB21L2 antagonizes the effects of BMP4 by repressing the BMP-induced gene expression. The nuclear protein binds SMAD1, the transducer of BMP2/4/7 signaling, but so far it is still unknown if MAB21L2 exerts its effects in a DNA-binding or a non-binding fashion. Our data of age- and osteoporosis-induced expression of MAB21L2 in hMSC made us hypothesize that BMP-signaling in stem cells is less effective in advanced age and even less so in primary osteoporosis due to transcriptional repression of BMPtarget genes. Despite high inter-individual variability in the gene expression level, as demonstrated in our heat map, we could validate the microarray results for both SOST and MAB21L2 in qPCR analysis with up to 13 different hMSC-OP and hMSC-old populations. We hereby demonstrate the reliability of our microarray approach, which was performed with a comparably low number of samples. Being inhibitors of WNT and BMP signaling, our two leading candidates are major hubs in blocking differentiation programs right at the beginning. Hereby, our data support the results of Rodriguez et al. and Dalle Carbonare et al., who demonstrated in vitro that osteoporotic hMSC exhibit diminished osteogenic differentiation potential. Future research will have to unravel how many of the genes differentially expressed in osteoporotic hMSC-OP are downstream SOST or MAB21L2 over-expression. Furthermore, we detected indications for osteoporotic stem cells actively WY 14643 enhancing osteoclastogenesis and therefore bone resorption.