Background/Purpose: Thy1 (CD90) is a glycosylated, glycophosphatidylinositol (GPI)-anchored membrane protein originally identified as a marker for mouse T cells but subsequent reports noted expression on stem cells, osteoblasts and fibroblasts. Thy1 is abundantly expressed by mesenchymal stem cells and inhibits adipogenesis. We examined the contribution of Thy1 to osteoblastogenesis and bone homeostasis.

Methods: A pre-osteoblastic MC3T3 cell line, and primary bone marrow derived mesenchymal stem cells from wild type (WT) C57BL/6 mice were cultured in osteogenic conditions for up to 21 days. Messenger RNA (mRNA) and protein expression of Thy1 and the OB differentiation markers RUNX2, BGLAP, COL1A1, were analyzed by real-time PCR and western blot. Similarly, mouse embryonic fibroblasts (MEFs) isolated from WT and Thy1 knockout (KO) mice were cultured in osteogenic media. Mineralized matrix and calcium deposition were examined with alizarin red staining. To study the impact of Thy1 depletion on bone quality in the normal and obese mice, 2 month old C57BL/6 WT and Thy1 KO mice were fed a normal or high fat diet for 2 months and long bones were analyzed with micro-CT to compare changes in bone architecture and overall bone quality.

Results: In vitro assays revealed that osteogenic conditions transiently increased Thy1 expression during OB differentiation. In vitro experiments with WT and Thy1 KO mesenchymal cells and MEFs further revealed that in the absence of Thy1, osteoblastogenesis was delayed as noted by alizarin red staining. Micro CT analysis of long bones in WT and Thy1 KO mice revealed that when Thy1 KO and WT mice were fed a high fat diet, bone quality at 4 months differed significantly between WT and KO mice. In particular, a significant reduction in trabecular bone quality was noted in Thy1 KO mice. The most significant differences were seen in tibial trabecular bone volume/total volume (BV/TV): 0.12 ± 0.02 in WT vs 0.09± 0.01 in Thy1 KO, (p < 0.03), femoral BV/TV: 0.37± 0.15 in WT vs. 0.19 ± 0.03 in Thy1 KO femur, (p < 0.03) and tibial trabecular bone connectivity density: 50.01± 10.85 in WT vs 28.29± 6.72 in Thy1 KO, (p <0.01), and femoral trabecular bone connectivity density: 42.03±14.42 in WT vs. 22.17±7.79 in Thy1 KO femur, (p <0.019); overall more than a 40% decrease. Interestingly, significant differences were not detected in WT and Thy1 KO mice fed a normal diet.

Conclusion: We find that Thy1 is required for differentiation of OB based on our in vitro experiments. Absence or decreased expression of Thy1 was associated with reduced bone quality. Of note, higher levels of Thy1 mRNA and protein levels were reported in osteoarthritis patients, a disease characterized by osteophyte formation with higher prevalence in obese individuals. Thus, Thy1 may serve as an important mechanistic link between bone formation and obesity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/thy1-is-a-positive-regulator-of-osteoblast-differentiation-and-modulates-bone-homeostasis-in-obese-mice/