Thymus-Derived PTH (TPTH) Is Increased after Thyroparathyroidectomy in C57BL6/Kalwrij Mice and Modulates Mouse Sensitivity to 5TGM1 Myeloma Cell Line

Abstract

We have previously reported a significant correlation between serum PTH levels and myeloma responses to proteasome inhibition and have recently demonstrated a crucial role for the parathyroid hormone receptor 1 (PTHR1) in vitro and in vivo. In this study the effect of abrogation of parathyroid-derived PTH was tested in 5TGM1 myeloma transplanted C57BL6/KaLwRij mice. To interrogate PTH activity in vivo, thyroparathyroidectomy (TX-PTX) was performed on the C57BL6/KaLwRij mice before or after the transplantation of 5TGM1 cells. Surgical success was confirmed by the rapid loss of measureable circulating PTH. In TX-PTX mice bearing transplanted 5TGM1 tumors, serum PTH levels was noted to recover 3-4 weeks after surgery and to achieve levels consistently higher than baseline levels pre TX-PTX. These data raised the compelling possibility of an alternative PTH source. To determine the potential source of the recovered PTH levels, multiple organs were examined. Whole tissue lysates were analyzed by Western blot and revealed a faint but specific band from thymic tissue, as expected with no obvious bands detected in 13 other tissues investigated including the liver, lung, kidney, ovary and testis. In addition, murine PTH mRNA transcripts from thymus RNA were measured by Real-Time PCR, using a specifically designed primer set that spans exons 1 and 2, since intron 1 is >1kb. These primers specifically discriminate mRNA transcripts from genomic DNA and improve the assay sensitivity for the detection of PTH transcripts. Using these custom-designed primers, control thymus expressed measureable PTH mRNA transcripts that was significantly elevated in TX-PTX mice. Since the thymus is considerably bigger than the parathyroid glands, PTH was immunoprecipitated before performing Western blot analysis. Following immunoprecipitation, a distinct PTH protein band was observed from the thymus in both TX-PTX and control mice.These data suggest that the thymus is the major source of serum PTH in TX-PTX mice and that the organ can compensate for the loss of serum PTH from the parathyroid gland. We propose that thymus-derived PTH in the TX-PTX mouse may contribute to the control of the 5TGM1 murine myeloma model. DisclosuresZangari:University of Arkansas for Medical Sciences: Employment; Novartis: Research Funding; Millennium: Research Funding; Onyx: Research Funding.