Erythropoietin Stimulates Bone Formation In Vivo By Targeting Both HSCs and MSCs

Previously we showed that HSCs isolated from stressed animals (an acute bleed) regulates osteoblast (OBs) differentiation from bone marrow stromal cells (BMSCs). This activity suggests that HSCs regulate the development of their own niche. The molecular basis for this activity is the production of BMP-2 and BMP-6 by HSCs. Yet what stimulates HSCs to produce BMPs is unknown. We hypothesized that erythropoietin (Epo) may activate HSCs to produce BMPs, or Epo directly activates BMSCs to form OBs. To test these possibilities, Epo serum levels in the bled vs. non-bleed mice were determined and found to match the time frame of HSC activation. Surprisingly, EpoR expression was identified on HSCs isolated using the SLAM-family of receptors, and its expression on HSCs increased in bled animals. In vitro stimulation with Epo activated the JAK-STAT signaling pathways, and importantly, BMP production by HSCs.

To determine if Epo directly acts on BMSCs, BMSC co-cultures were established with HSCs derived from bled (B-HSC) or non-bled (NB-HSCs) animals, or treated with rhEpo alone for 14d. Differentiation along the OB lineage was evaluated for CFU-fibroblastic and osteoblastic (CFU-F, CFU-OB) formation, QRT-PCR for Runx2 (OB-specific transcription factor), bone sialoprotein and osteocalcin, or for bone mineral formation (alizarin red staining). BMSCs responded to the presence of NB-HSCs by increasing their generation of CFU-OB and CFU-F compared to no HSCs. Co-culture with B-HSCs or rhEpo treatments were significantly better able to induce CFU-OB and CFU-F differentiation of the BMSCs. QRT-PCR demonstrates that the OB-specific transcripts were increased with rhEpo or co-culture with B-HSC compared to NB-HSC or vehicle. Alizarine red staining also demonstrated an increase in mineral apposition in response to Epo or B-HSC.

To directly determine if Epo stimulates bone formation in vivo, newborn mice were treated with rhEpo (1,500U-6,000U/kg, 3x/week), anabolic PTH (as a positive control) or vehicle for 4 weeks. rhEpo increased the hematocrit and hemoglobin levels in a dose dependant manner. As expected, PTH increased the bone formation. rhEpo treatments enhanced OB numbers in long bone sections, and micro-CT measurements of the vertebra revealed a significant increase in bone formation parameters including bone mineral density (BMD), bone volume fraction (BVF) and trabecular number (Tb.N.).

These data for the first time demonstrate that Epo regulates the formation of the HSC niche by both direct and indirect pathways, and further demonstrates a coupling between hematopoesis and osteopoiesis in the marrow. These results also suggest that targeting the Epo/EpoR pathway may serve as a therapeutic modality to treat skeletal or mesenchymal abnormalities in humans.