Development of a Technique for Introduction of an Expressed cDNA into Parathyroid Cells by Direct Injection

PTH is a major mediator of bone and mineral metabolism. However, physiological and pathological investigations of parathyroid cells (PTCs) have been limited because of the lack of available cell lines and because the organ is too small for detailed studies. Here, we describe a novel method for adenovirus-mediated cDNA transfer into PTCs, and we show the accuracy of the method in a rat model of uremia-induced secondary hyperparathyroidism. Rats underwent a 5/6-nephrectomy and were fed with a high-phosphate diet for 8 wk. The parathyroid glands were surgically exposed and adenoviruses containing LacZ or Ca-sensing receptor (CaSR) were directly injected into the glands under a zoom-stereo microscope. The parathyroid glands were analyzed for infection of adenovirus and immunohistochemically for expression of CaSR. The functional activity of exogenous CaSR in PTCs after this treatment was investigated based on changes of the calcium and PTH curve. A virus concentration of more than 10 plaque-forming units/ml was required for adequate infection of PTCs within 7 d after treatment. Marked increase of CaSR-positive PTCs by 2.39 0.72 times relative to control treatment, and significant colocalization of CaSR overexpression and virus labeling, were observed in glands after gene introduction. The calcium and PTH curve was shifted to the left from the basal position (set point, 1.10 0.09 to 0.76 0.12 mM; P 0.0001), indicating successful introduction of a functionally active gene into the PTCs. This technique may facilitate an elucidation of biological effects through targeting and identification of specific features of PTCs, which may provide the basis for new clinical approaches. Effects of a Novel Selective Androgen Receptor Modulator on DexamethasoneInduced and Hypogonadism-Induced Muscle Atrophy Amanda Jones, Dong-Jin Hwang, Ramesh Narayanan, Duane D. Miller, and James T. Dalton (Endocrinology, published June 9, 2010, 10.1210/en.2010-0150) ABSTRACT Glucocorticoids are the most widely used antiinflammatory drugs in the world. However, prolonged use of glucocorticoids results in undesirable side effects such as muscle wasting, osteoporosis, and diabetes. Skeletal muscle wasting, which currently has no approved therapy, is a debilitating condition resulting from either reduced muscle protein synthesis or increased degradation. The imbalance in protein synthesis could occur from increased expression and function of muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx)/atrogin and muscle ring finger 1 (MuRF1), or decreased function of the IGF-I and phosphatidylinositol-3 kinase/Akt kinase pathways. We examined the effects of a nonsteroidal tissue selective androgen receptor modulator (SARM) and testosterone on glucocorticoid-induced muscle atrophy and castrationinduced muscle atrophy. The SARM and testosterone propionate blocked the dexamethasone-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including Forkhead box O. Dexamethasone caused a significant up-regulation in the expression of ubiquitin ligases, but testosterone propionate and SARM administration blocked this effect by phosphorylating Forkhead box O. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of MAFbx and MuRF1 and down-regulation of IGF-I, all of which was attenuated by a SARM. The results suggest that levator ani atrophy caused by hypogonadism may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on up-regulation of MAFbx and MuRF1. Our studies provide the first evidence that glucocorticoidand hypogonadism-induced muscle atrophy are mediated by distinct but overlapping mechanisms and that SARMs may provide a more effective and selective pharmacological approach to prevent glucocorticoid-induced muscle loss than steroidal androgen therapy.Glucocorticoids are the most widely used antiinflammatory drugs in the world. However, prolonged use of glucocorticoids results in undesirable side effects such as muscle wasting, osteoporosis, and diabetes. Skeletal muscle wasting, which currently has no approved therapy, is a debilitating condition resulting from either reduced muscle protein synthesis or increased degradation. The imbalance in protein synthesis could occur from increased expression and function of muscle-specific ubiquitin ligases, muscle atrophy F-box (MAFbx)/atrogin and muscle ring finger 1 (MuRF1), or decreased function of the IGF-I and phosphatidylinositol-3 kinase/Akt kinase pathways. We examined the effects of a nonsteroidal tissue selective androgen receptor modulator (SARM) and testosterone on glucocorticoid-induced muscle atrophy and castrationinduced muscle atrophy. The SARM and testosterone propionate blocked the dexamethasone-induced dephosphorylation of Akt and other proteins involved in protein synthesis, including Forkhead box O. Dexamethasone caused a significant up-regulation in the expression of ubiquitin ligases, but testosterone propionate and SARM administration blocked this effect by phosphorylating Forkhead box O. Castration induced rapid myopathy of the levator ani muscle, accompanied by up-regulation of MAFbx and MuRF1 and down-regulation of IGF-I, all of which was attenuated by a SARM. The results suggest that levator ani atrophy caused by hypogonadism may be the result of loss of IGF-I stimulation, whereas that caused by glucocorticoid treatment relies almost solely on up-regulation of MAFbx and MuRF1. Our studies provide the first evidence that glucocorticoidand hypogonadism-induced muscle atrophy are mediated by distinct but overlapping mechanisms and that SARMs may provide a more effective and selective pharmacological approach to prevent glucocorticoid-induced muscle loss than steroidal androgen therapy. Aberrant Expression and Modification of Silencing Mediator of Retinoic acid and Thyroid Hormone Receptors Involved in the Pathogenesis of Tumoral Cortisol Resistance Jingjing Jiang, Na Li, Xiaolin Wang, Yan Lu, Yufang Bi, Weiqing Wang, Xiaoying Li, and Guang Ning (Endocrinology, published June 16, 2010, 10.1210/en.2010-0335) ABSTRACT Ectopic ACTH syndrome (EAS) accounts for 10-15% of cases of Cushing’s syndrome and is mostly caused by small cell lung cancers or thymic carcinoids. EAS is characterized by tumoral cortisol resistance, whose underlying mechanism remains unknown. In this study, we reported that silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a major nuclear corepressor, was aberrantly expressed in ACTH-secreting thymic carcinoids. Overexpression and knockdown of SMRT in the ACTHsecreting AtT-20 cell line demonstrated that SMRT participated in the negative feedback of dexamethasone-mediated suppression of proopiomelanocortin. Posttranslational modification by the small ubiquitin-like modifiers (SUMO), i.e. SUMOylation plays an important role in fine-tuning transcriptional activities. SUMOylation of SMRT 3560 Abstracts Translational Highlights from JCEM J Clin Endocrinol Metab, July 2010, 95(7):3558-3562Ectopic ACTH syndrome (EAS) accounts for 10-15% of cases of Cushing’s syndrome and is mostly caused by small cell lung cancers or thymic carcinoids. EAS is characterized by tumoral cortisol resistance, whose underlying mechanism remains unknown. In this study, we reported that silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a major nuclear corepressor, was aberrantly expressed in ACTH-secreting thymic carcinoids. Overexpression and knockdown of SMRT in the ACTHsecreting AtT-20 cell line demonstrated that SMRT participated in the negative feedback of dexamethasone-mediated suppression of proopiomelanocortin. Posttranslational modification by the small ubiquitin-like modifiers (SUMO), i.e. SUMOylation plays an important role in fine-tuning transcriptional activities. SUMOylation of SMRT 3560 Abstracts Translational Highlights from JCEM J Clin Endocrinol Metab, July 2010, 95(7):3558-3562