Abstract
Primary hyperparathyroidism (PHPT) is a common endocrinopathy characterized by hypercalcemia and elevated levels of parathyroid hormone. The primary cause of PHPT is a benign overgrowth of parathyroid tissue causing excessive secretion of parathyroid hormone. However, the molecular etiology of PHPT is incompletely defined. Here, we demonstrate that semaphorin3d (Sema3d), a secreted glycoprotein, is expressed in the developing parathyroid gland in mice. We also observed that genetic deletion of Sema3d leads to parathyroid hyperplasia, causing PHPT. In vivo and in vitro experiments using histology, immunohistochemistry, biochemical, RT-qPCR, and immunoblotting assays revealed that Sema3d inhibits parathyroid cell proliferation by decreasing the epidermal growth factor receptor (EGFR)/Erb-B2 receptor tyrosine kinase (ERBB) signaling pathway. We further demonstrate that EGFR signaling is elevated in Sema3d-/- parathyroid glands and that pharmacological inhibition of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype. We propose that because Sema3d is a secreted protein, it may be possible to use recombinant Sema3d or derived peptides to inhibit parathyroid cell proliferation causing hyperplasia and hyperparathyroidism. Collectively, these findings identify Sema3d as a negative regulator of parathyroid growth.
Highlights
Primary hyperparathyroidism (PHPT) is a common endocrinopathy characterized by hypercalcemia and elevated levels of parathyroid hormone
In vivo and in vitro experiments using histology, immunohistochemistry, biochemical, RT-qPCR, and immunoblotting assays revealed that Sema3d inhibits parathyroid cell proliferation by decreasing the epidermal growth factor receptor (EGFR)/Erb-B2 receptor tyrosine kinase (ERBB) signaling pathway
We further demonstrate that EGFR signaling is elevated in Sema3d؊/؊ parathyroid glands and that pharmacological inhibition of EGFR signaling can partially rescue the parathyroid hyperplasia phenotype
Summary
To determine the expression of Sema3d during parathyroid development, we performed expression and lineage tracing analyses in Sema3dGFPCre/ϩ mice [30]. To determine the molecular changes associated with increased parathyroid cell proliferation and hyperplasia in Sema3dϪ/Ϫ mice, we analyzed the expression of cell cycle regulators, including cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitor (CDKIs) by qPCR on RNA isolated from microdissected parathyroid glands. No significant changes were observed (Fig. S2) Together, these results suggest that Sema3d inhibits parathyroid cell proliferation in vivo by regulating the expression of cell cycle genes. The addition of Sema3d significantly decreased (ϳ30%, p Ͻ 0.001) the number of Ki-67–positive cells induced by NSC 228155 treatment This suggests that Sema3d regulates cell cycle progression by signaling through the EGFR (Fig. 5C). PTH levels were significantly reduced after erlotinib treatment (Fig. 6D) Together, these results indicate that blocking EGFR signaling could partially rescue the hyperplasia and hyperparathyroidism phenotype seen in Sema3dϪ/Ϫ mice
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