Role of the Parathyroid Hormone Type 1 Receptor (PTH1R) as a Mechanosensor in Osteocyte Survival

Abstract

Osteocytes have a major role in the control of bone remodeling. Mechanical stimulation decreases osteocyte apoptosis and promotes bone accrual, whereas skeletal unloading is deleterious in both respects. PTH1R ablation or overexpression in osteocytes in mice produces trabecular bone loss or increases bone mass, respectively. The latter effect was related to a decreased osteocyte apoptosis. Here, the putative role of PTH1R activation in osteocyte protection conferred by mechanical stimulation was assessed. Osteocytic MLO-Y4 cells were subjected to mechanical stimuli represented by hypotonic shock (216 mOsm/kg) or pulsatile fluid flow (8 Hz, 10 dynes/cm(2)) for a short pulse (10 min), with or without PTH1R antagonists or after transfection with specific PTHrP or PTH1R siRNA. These mechanical stimuli prevented cell death induced within 6 hours by etoposide (50 μM), related to PTHrP overexpression; and this effect was abolished by the calcium antagonist verapamil (1 μM), a phospholipase C (PLC) inhibitor (U73122; 10 μM), and a PKA activation inhibitor, Rp-cAMPS (25 μM), in these cells. Each mechanical stimulus also rapidly induced β-catenin stabilization and nuclear ERK translocation, which were inhibited by the PTH1R antagonist PTHrP(7-34) (1 μM), or PTH1R siRNA, and mimicked by PTHrP(1-36) (100 nM). Mechanical stretching by hypotonic shock did not affect cAMP production but rapidly (<1 min) stimulated Ca(i)(2+) transients in PTH1R-overexpressing HEK-293 cells and in MLO-Y4 cells, in which calcium signaling was unaffected by the presence of a PTHrP antiserum or PTHrP siRNA but inhibited by knocking down PTH1R. These novel findings indicate that PTH1R is an important component of mechanical signal transduction in osteocytic MLO-Y4 cells, and that PTH1R activation by PTHrP-independent and dependent mechanisms has a relevant role in the prosurvival action of mechanical stimulus in these cells.

Keywords: MECHANICAL STIMULUS; MLO-Y4 CELLS; OSTEOCYTE SURVIVAL; PTH TYPE 1 RECEPTOR; PTHRP.