Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism

Abstract

Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy. Additionally, we inquired whether Nmp4 interferes with anticatabolic efficacy. Wild-type and Nmp4-/- mice were ovariectomized at 12 weeks of age, followed by therapy regimens, administered from 16 to 24 weeks, and included individually or combined PTH, alendronate (ALN), zoledronate (ZOL), and raloxifene (RAL). Anabolic therapeutic efficacy generally corresponded with PTH + RAL = PTH + ZOL > PTH + ALN = PTH > vehicle control. Loss of Nmp4 enhanced femoral trabecular bone increases under PTH + RAL and PTH + ZOL. RAL and ZOL promoted bone restoration, but unexpectedly, loss of Nmp4 boosted RAL-induced increases in femoral trabecular bone. The combination of PTH, RAL, and loss of Nmp4 significantly increased bone marrow osteoprogenitor number, but did not affect adipogenesis or osteoclastogenesis. RAL, but not ZOL, increased osteoprogenitors in both genotypes. Nmp4 status did not influence bone serum marker responses to treatments, but Nmp4-/- mice as a group showed elevated levels of the bone formation marker osteocalcin. We conclude that the heightened osteoanabolism of the Nmp4-/- skeleton enhances the effectiveness of diverse osteoporosis treatments, in part by increasing hyperanabolic osteoprogenitors. Nmp4 provides a promising target pathway for identifying barriers to pharmacologically induced bone formation.

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality

“…However loss of Nmp4 did significantly impact some aspects of femoral cortical geometry, such as cortical thickness, marrow area as well as other related parameters (Table 3). Altogether, these results are similar to the data we reported in older ovariectomized mice (95).…”

“…Moreover, preliminary experiments with shRNA knockdown of Nmp4 in MC3T3-E1 cells yielded a similar Collagen/well vs. Cells/well profile (data not shown). This is consistent with our previous in vivo data showing that the Nmp4 -/mice harbor more bone marrow osteoprogenitors than WT, which in turn produce more bone when stimulated (16,41,95). We conclude that loss of Nmp4 converts osteoprogenitors/osteoblasts into super-secretors of bone matrix while moderately enhancing their proliferative activity.…”

“…RAL is normally administered as a 60mg daily dose, therefore based on a 60kg patient the quantity would be 1 mg/kg/day. The assumption is 100% absorption therefore the full dose was administered as a subcutaneous injection (95). Our euthanasia protocol involves using carbon dioxide inhalation at 20%V/min followed by bilateral pneumothorax or cervical dislocation in compliance with the guidelines of our Animal Care and Use Committee.…”

“…We reported that the transcription factor Nuclear Matrix Protein 4 (Nmp4, Zfp384, Ciz, ZNF384) suppresses the action of osteoanabolics (15,16,41,70,90,95) and thus elucidation of the upstream and downstream effectors in the Nmp4 pathway may provide a map of the innate barriers to PTH-induced bone formation. Indeed, as a trans-acting protein Nmp4 is well positioned to control multiple aspects of bone formation.…”

Efficacy of targeting bone-specific GIP receptor in ovariectomy-induced bone loss

“…Thick cross-sections (500 µm-thick) at the mid-diaphysis were cut with a low speed precision saw and incubated in saline or 0.1 M acetic acid (pH 4.5) for 24 h. The resulting solution was buffered with 1 M Tris and fluorescence readings (OVX + Aln, n = 8). These doses and regimens of GIP analogues and alendronate were based on previous published studies where these molecules were proven active with beneficial effects on bone or equivalent to approved clinical dose (Mabilleau et al 2014, Shao et al 2017. Eight sham-operated female BALB/c mice with the same age and injected daily with saline were used as controls (Sham + Veh).…”