Bone Abstracts

Thalassemia is a chronic anemia associate with high rates of osteoporosis. To determine how erythropoiesis leads to bone loss in thalassemia, we used the th3/+ thalassemia mouse to study the role of erythropoietin (EPO) and hematopoietic progenitors (HP), since they are both increased in thalassemia.

Methods: Bone marrow mesenchymal stem cells (MSCs) cultures and MSC cocultures with various HP from wild type (wt) and th3/+ mice were differentiated into osteoblasts. Osteogenesis was determined by colony forming units for Osteoblasts (CFU-O) and qPCR for osteogenesis genes (Runx2, BSP, osterix, and osteocalcin).

Results: Compared to wt, bone formation was decreased in th3/+ animals. MSC cultures from th3/+ showed decreased osteogenesis, indicating that altered MSC fate leads to decreased osteogenesis in thalassemia. Treatment of MSC cultures with EPO antibody (EPO-Ab) or soluble EPO receptor (sEPOR) reversed the decreased osteogenesis in th3/, demonstrating that EPO signaling guides MSC fate. Furthermore, increased EPO and EPOR expression and changes in JAK2/Stat signaling were seen in thalassemia MSC.

We then isolated various HP and co-cultured them with MSC from wt animals. Lin-Sca+ ckit+ (LSK) precursors from th3/+ mice led to decreased osteogenesis, while mature erythroid cells (Ter119+) had no effect. Treatment of LSK–MSC co-cultures with EPO-Ab or sEPOR normalized the suppressed osteogenesis in th3/+, indicating a role of EPO signaling in LSK–MSK interactions. Consistently, EPOR and EPO expression in thalassemia LSK was increased.

Bone morphogenic proteins (BMPs) potentiate both erythroid expansion and osteogenesis. BMP-2, -4,and -6 expression in LSK was decreased in th3/+ compared to wt, and reversed with treatment with EPO-Ab or JAK2 inhibitor. In LSK–MSC cocultures, the effect of EPO-Ab on osteogenesis was lost with additional treatment with noggin, a BMP inhibitor, suggesting that EPO affects LSK–MSC interaction in thalassemia by regulating BMP expression in LSK.

Overall, in th3/+ thalassemia mice, we observed i) decreased osteogenesis as a result of altered MSC differentiation. ii) EPO produced by thalassemic MSC decreases osteogenesis via direct EPO signaling, supporting an extra-hematopoietic action of EPO. iii) LSK but not mature erythroid cells interact with MSCs in th3/+ and decrease osteogenesis iii). EPO guides MSC–LSK interactions by regulating BMP expression in hematopoietic progenitors.