SAT 558 Tsh Modulation Of Bone Biology Further Evidence From A Recombinant Tsh b Variant.
Abstract We have previously shown that TSH has modulatory influences on osteoblasts and osteoclasts and can act as a bone protection ligand. We have also shown that a novel TSH b variant protein is produced locally by murine and human bone marrow macrophages (Endocrinology 154:4919 26, 2013 and Endocrinology 157:3658 67, 2016) Furthermore, we have also shown by molecular modelling that both TSH b and TSH bv can bind to the native TSH receptor. In order to characterize the biological activity of this TSH b variant we have inserted the gene sequence together with a FLAG tag into the P secTag2 vector which was then cloned into mammalian HEK 293 cells. Western blot analysis identified 17KD and a 12KD proteins corresponding to the wild type TSH b and the TSH bv nucleotide sequence in cell culture lysates and, less so in supernatants, by using a FLAG monoclonal antibody and a TSH b polyclonal antibody. Protein bands of ~34 KD and 24 KD were observed only in the absence of DTT suggesting dimeric forms of TSH b and TSH bv respectively. Detection by Coomassie staining followed by sequence analysis using mass spectrometry confirmed the identity of these proteins after column purification. Furthermore, cyclic AMP responses were generated by both supernatant preparations with an 8 fold increase in luciferase activity with TSH b and a 4 fold increase with TSH bv. These data demonstrate the inherent bioactivity of single TSH b chains which was mirrored by TSH bv. Local paracrine action by macrophage derived TSH bv on osteoblasts and osteoclasts is, therefore, most likely and raises the possibility of this variant form having significant osteoprotective activity.
Journal of the Endocrine Society