To further examine the influence of microgravity mechanical
To further examine the influence of microgravity mechanical unloading on differentiation of mesenchymal and hematopoietic lineages, we investigated genes associated with terminal differentiation of these lineages. We found a broad down-regulation of early to mid stage differentiation markers and down-regulation or no alteration in terminal osteoclast and osteoblast markers. Specifically, we found alterations in three osteoclast differentiation markers: IL-6, MITF, and CSF2 (GM-CSF). IL-6, involved in bone homeostasis, acts together with the IL-6 receptor (IL-6R) to induce the apexbio calculator of RANKL on the surface of osteoblasts, enabling interaction with RANK on the surface of osteoclast progenitors (Yoshitake et al., 2008). This interaction allows the differentiation of osteoclast progenitors into mature osteoclasts (Yoshitake et al., 2008). However, IL-6 can also act directly on osteoclast progenitors to suppress their differentiation and facilitate proliferation through the up-regulation of RANKL in osteoblasts (Kwan Tat et al., 2004). Although we did not investigate the expression of IL-6R in bone marrow through PCR, we found no difference in expression in Affymetrix results (data not shown), and in conjunction with our other gene expression data these results suggest suppression of osteoclast and osteoblast differentiation rather than promotion of osteoblast differentiation or osteoclast-inducing activities. MITF, which is activated by RANKL and also plays a critical role in osteoclastogenesis, was down-regulated. Mutations in this gene profoundly affect the osteoclast lineage, and through these mutation studies it was found that MITF transcriptionally regulates the expression of many osteoclast genes including CLCN7, CTSK, OSCAR, OSTM1, and TRAP (Lu et al., 2010). CSF2, however, stimulates the fusion of mono-nuclear osteoclasts into bone resorbing, multi-nucleated osteoclasts by inducing the expression of DC-STAMP, a transmembrane protein that is primarily expressed in dendritic cells, but also plays a role in osteoclastogenesis. CSF2 has been shown to suppress RANKL mediated differentiation of osteoclast precursors by inhibiting c-FOS, however, it also plays a critical role in osteoclastic bone erosion. Expression of CSF2 at an early stage of osteoclast differentiation causes the suppression of osteoclast precursor differentiation through negative regulation of RANKL mediated osteoclast differentiation (Lee et al., 2009), suggesting that CSF2 down-regulation results in inactivation of NFATc1-induced expression of DC-STAMP and consequently fusion of osteoprecursors into mature, multi-nucleated and fully functional bone resorbing cells. However, CSF2 also stimulates the differentiation of a number of blood cell lineages, including basophils, neutrophils, eosinophils, erythrocytes and megakaryocytes, indicating that its down-regulation may impact the differentiation of a number of cells within the bone marrow compartment. We found no statistically significant alterations in a number of other genes expressed in terminally differentiated osteoclasts, including ACP5, CALCR, CTSK, CSF1, NFκB1, TNFSF11, TRAF6, TNF, and MYC. As terminally differentiated osteoclasts express elevated levels of these differentiation markers, and despite the activation of bone resorption, these results suggest that differentiation of osteoclasts in microgravity may also have been partly inhibited. We also investigated terminal differentiation markers of lineages derived from MSCs and found down-regulation of most genes associated with osteogenesis, tenogenesis, adipogenesis, and chondrogenesis. Specifically, we found down-regulation of IL-1β, which is a potent activator of osteoclastogenesis and therefore enhances bone resorption and inhibits bone formation and chondrocyte production (Nguyen et al., 1991; Boyle et al., 2003). Down-regulation of this gene may indicate impairment in the normal osteoclastogenesis activation pathways, consistent with our gene expression results. IL-1β also down-regulates the expression of SOX9, one of the major inducers of chondrocyte differentiation, through a mechanism mediated by NFκB (Majumdar et al., 2001). However, we found no alteration in the expression of SOX9, indicating that chondrocyte differentiation is not increased in microgravity. Furthermore, BMP2 expression, which overrides IL-1β induced suppression of SOX9 by inhibiting NFκB activity, was not altered, providing further evidence that chondrocyte differentiation was not enhanced in microgravity via IL-1β down-regulation. We also found no alteration in other genes investigated, which are known to affect chondrogenesis, including BMP4 and BMP6, GDF5, GDF6 and GDF7, HAT1, ITGαx, ABCB1a, and KAT2B. The trend of decreased differentiation in chondrocytes was also observed in the genes involved in adipogenesis (PPARγ), whilst no change was seen in myogenesis differentiation (JAG1) (Farmer, 2005). Gene expression analyses of several terminal differentiation markers for osteoblasts or osteocytes were found to have no significant alterations, including COL1A1, FGF2, BGLAP1, and FGF23, whilst the osteocyte marker, GDF15 was down-regulated. Furthermore, osteoblastogenesis differentiation inducers were found to be down-regulated, including HDAC1, BMP7, TGFβ1 and PTK2, whilst other inducers, BMP2, FGF10, and RUNX2, were found to have no alteration, providing a picture of broad down-regulation of mesenchymal stem cell differentiation in microgravity.