The only established function of thyroid stimulating hormone (TSH or thyrotropin) is to stimulate the synthesis and secretion of thyroid hormone, the overproduction of which results in osteoporosis. To examine whether TSH directly regulates bone remodeling, we generated a TSHR null mouse by replacing exon 1 of the TSHR gene with green fluorescent protein (GFP). We first established that TSHRs were present in osteoclast and osteoblast precursors. TSHR null, and more importantly, heterozygous mice were both profoundly osteoporotic. Additionally, they exhibited focal pagetoid osteosclerosis reminiscent of disordered bone remodeling. Ex vivo hematopoietic stem cell cultures from both TSHR-/- and TSHR mice showed marked increases in TRAP (tartrate resistant acid phosphatase)-positive osteoclast formation and osteoclast markers in the presence of RANK-L (receptor activator for NF(B-ligand). Likewise, recombinant human TSH inhibited RANK-L-induced TRAP-positive osteoclast formation. TSHR overexpression in osteoclast progenitors (RAW264.7) also resulted in increased apoptosis. Taken together, the results provided compelling evidence that TSH inhibited the formation and lifespan of osteoclasts, and that this inhibition was mediated via the RANK-L signaling pathway. Thus, in Specific Aim 1, we will further investigate the effects of abrogating and overexpressing the TSHR on osteoclast formation, lifespan, and function. We will first examine the stage-specific expression of TSHRs in osteoclast precursors during osteoclastogenesis. We will then study the consequences of the overexpression of TSHR and a constitutively active mutant, Val509AIa-TSHR, on osteoclast formation, lifespan, and bone resorption. Next, we will examine increased osteoclast formation and bone resorption is due to increased TNFalpha and IL-7 production. Finally, we will study the consequences of TSHR and Val509AIa-TSHR overexpression in vivo in transgenic mice using the TRAP promoter to drive expression selectively in osteoclasts.
In Specific Aim 2, we will investigate the detailed mechanism of the anti-osteoclastogenic action of TSH, because TSH blocks RANK-L-induced osteoclastogenesis and JNK and NF(B activation in RAW-C3 cells, and a deficiency of TSHR increases these parameters in primary osteoclasts. We shall determine if the inhibition is reversed by overexpression of JNK and/or IKK in RAW-C3 cells and in primary cells, and if inhibition of osteoclast formation by TSH is through the TRAFs (tumor necrosis factor receptor-associated factors) signaling, upstream from JNK and NF(B signaling. Next, we will investigate if enhanced JNK and IkappaBalpha phosphorylation in osteoclast precursors from TSHR-/-mice could be normalized by TSHR or Val509AIa-TSHR overexpression as well as dominant forms of JNK, IKK or TRAFs overexpression. Overall, these studies will characterize a unique role of the extrathyroidal TSHR in osteoclast formation, function and its mechanism.
