Modeling vitamin D insufficiency and moderate deficiency in adult mice via dietary cholecalciferol restriction.

PURPOSE: We sought to develop and characterize a model of human vitamin D nutritional insufficiency/deficiency in the adult mouse, which could have broad utility in examining health consequences of this common condition. METHODS: Adult mice were fed diets containing cholecalciferol contents of 0.05 IU/g, 0.25 IU/g, 0.5 IU/g or 1.5 IU/g for four months. We studied induction of steady-state vitamin D insufficiency, and its consequences on primary cholecalciferol metabolite levels, calcium homeostasis, parathyroid physiology, and bone morphology. RESULTS: All diets were well tolerated, without adverse effects on body weight. Diets containing 0.05 IU/g and 0.25 IU/g cholecalciferol significantly lowered serum 25-hydroxyvitamin D levels (median 25OHD, 10.5 ng/ml, and 21.6 ng/ml, respectively), starting as early as one month following initiation of the diets, maintained through the four-month experimental period. The 0.05 IU/g diet significantly decreased 1,25-dihydroxyvitamin D (1,25OH2D) levels (median, 78 pg/ml). Despite these decreased 25OHD and 1,25OH2D levels, the diets did not alter parathyroid gland morphology or parathyroid cell proliferation. There were no statistical differences in the serum total calcium and serum PTH levels among the various dietary groups. Furthermore, the 0.05 IU/g diet did not cause any alterations in the cortical and trabecular bone morphology, as determined by microCT. CONCLUSIONS: The dietary manipulations yielded states of vitamin D insufficiency or modest deficiency in adult mice, with no overtly detectable impact on parathyroid and bone physiology, and calcium homeostasis. This model system may be of value to study health effects of vitamin D insufficiency/deficiency especially on extraskeletal phenotypes such as cancer susceptibility or immune function.

Influence of Vitamin D Deficiency on Cyclin D1-Induced Parathyroid Tumorigenesis.

Primary hyperparathyroidism (PHPT) is a common endocrinopathy for which several pathogenic mechanisms, including cyclin D1 overexpression, have been identified. Vitamin D nutritional status may influence parathyroid tumorigenesis, but evidence remains circumstantial. To assess the potential influence of vitamin D insufficiency/deficiency on initiation or progression of parathyroid tumorigenesis, we superimposed vitamin D insufficiency or deficiency on parathyroid tumor-prone parathyroid hormone-cyclin D1 transgenic mice. Mice were placed on diets containing either 2.75 IU/g, 0.25 IU/g, or 0.05 IU/g cholecalciferol, either prior to expected onset of PHPT or after onset of biochemical PHPT. When introduced early, superimposed vitamin D insufficiency/deficiency had no effect on serum calcium or on parathyroid gland growth. However, when introduced after the onset of biochemical PHPT, vitamin D deficiency led to larger parathyroid glands without differences in serum biochemical parameters. Our results suggest that low vitamin D status enhances proliferation of parathyroid cells whose growth is already being tumorigenically driven, in contrast to its apparent lack of direct proliferation-initiating action on normally growing parathyroid cells in this model. These results are consistent with the hypothesis that suboptimal vitamin D status may not increase incidence of de novo parathyroid tumorigenesis but may accelerate growth of a preexisting parathyroid tumor.

CDK4/6 Dependence of Cyclin D1-Driven Parathyroid Neoplasia in Transgenic Mice.

The protein product of the cyclin D1 oncogene functions by activating partner cyclin-dependent kinases (cdk)4 or cdk6 to phosphorylate, thereby inactivating, the retinoblastoma protein pRB. Nonclassical, cdk-independent, functions of cyclin D1 have been described but their role in cyclin D1-driven neoplasia, with attendant implications for recently approved cdk4/6 chemotherapeutic inhibitors, requires further examination. We investigated whether cyclin D1's role in parathyroid tumorigenesis in vivo is effected primarily through kinase-dependent or kinase-independent mechanisms. Using a mouse model of cyclin D1-driven parathyroid tumorigenesis (PTH-D1), we generated new transgenic lines harboring a mutant cyclin D1 (KE) that is unable to activate its partner kinases. While this kinase-dead KE mutant effectively drove mammary tumorigenesis in an analogous model, parathyroid-overexpressed cyclin D1 KE mice did not develop the characteristic biochemical hyperparathyroidism or parathyroid hypercellularity of PTH-D1 mice. These results strongly suggest that in parathyroid cells, cyclin D1 drives tumorigenesis predominantly through cdk-dependent mechanisms, in marked contrast with the cdk-independence of cyclin D1-driven mouse mammary cancer. These findings highlight crucial tissue-specific mechanistic differences in cyclin D1-driven tumorigenesis, suggest that parathyroid/endocrine cells may be more tumorigenically vulnerable to acquired genetic perturbations in cdk-mediated proliferative control than other tissues, and carry important considerations for therapeutic intervention.

Cyclin D1 overexpression increases susceptibility to 4-nitroquinoline-1-oxide-induced dysplasia and neoplasia in murine squamous oral epithelium.

The cyclin D1 oncogene is frequently amplified/overexpressed in oral squamous cell carcinomas. Mice with overexpression of cyclin D1 targeted to the stratified squamous epithelia of the tongue, esophagus, and forestomach develop a phenotype of epithelial dysplasia at these sites. In this study, we examined the effect of cyclin D1 overexpression on susceptibility of mice to carcinogen-induced tumorigenesis, using 4-nitroquinoline-1-oxide (4NQO), an established potent oral carcinogen in mice. Cyclin D1 overexpressing mice and nontransgenic littermates were administered 4NQO (20 or 50 parts per million (ppm) in the drinking water) for 8 wk and monitored for an additional 16 wk. Histopathological analyses of the tongue revealed significantly higher severity of dysplasia in the cyclin D1 overexpression mice, compared with nontransgenic controls and with untreated controls. Moreover, only the cyclin D1 overexpression mice developed neoplastic lesions in the oro-esophageal epithelia. Examination of the dysplastic and neoplastic lesions revealed abnormal proliferation. Our findings suggest that cyclin D1 overexpression enhances susceptibility to carcinogen-induced oral tumorigenesis. These results underscore the importance of cyclin D1 in the process of oral neoplastic development. Further, they emphasize the value of this transgenic model to study the pathogenesis of oral precancer and cancer and establish it as a model system to test candidate agents for chemoprevention of upper aero-digestive cancer.

Abnormal parathyroid cell proliferation precedes biochemical abnormalities in a mouse model of primary hyperparathyroidism.

The properties of neoplastic proliferation and hormonal dysregulation are tightly linked in primary hyperparathyroidism (HPT). However, whether abnormal parathyroid proliferation is the cause or result of a shift in calcium-sensitive parathyroid hormonal regulation has been controversial. We addressed this issue by analyzing the temporal sequence of these fundamental abnormalities in a mouse model of primary HPT. These transgenic mice (PTH-D1) harbor a transgene that targets overexpression of the cyclin D1 oncogene to parathyroid cells, resulting in parathyroid hypercellularity with a phenotype of chronic biochemical HPT and, notably, an abnormal in vivo PTH-calcium set point. We examined parathyroid cell proliferation and biochemical alterations in PTH-D1 and control wild-type mice from ages 1-14 months. Strikingly, abnormal parathyroid proliferation regularly preceded dysregulation of the calcium-PTH axis, supporting the concept that disturbed parathyroid proliferation is the crucial primary initiator leading to the development of the biochemical phenotype of HPT. Furthermore, we observed that decreased expression of the calcium-sensing receptor in the parathyroid glands occurs several months before development of biochemical HPT, suggesting that decreased calcium-sensing receptor may not be sufficient to cause PTH dysregulation in this animal model of primary HPT.

Molecular pathogenesis of primary hyperparathyroidism.

This article will primarily focus on the molecular pathogenesis of common, sporadic (nonfamilial) parathyroid adenomas; two genes currently have established roles in the development of these tumors. The cyclin D1/PRAD1 gene was identified as a clonally activated oncogene in parathyroid adenomas and has subsequently been established as a major contributor to human neoplasia. Overexpression of cyclin D1, a key regulator of the cell cycle, has been implicated in the pathogenesis of 20-40% of sporadic parathyroid adenomas. That such cyclin D1 overexpression indeed constitutes a stimulus to excessive parathyroid cell proliferation has been confirmed experimentally by the development of a transgenic mouse model with parathyroid-targeted overexpression of cyclin D1. Parathyroid hormone (PTH)-cyclin D1 transgenic mice develop parathyroid hypercellularity, biochemical hyperparathyroidism, and a shifted in vivo parathyroid-calcium setpoint; these mice constitute an animal model of human hyperparathyroidism in which aspects of tumorigenesis, parathyroid secretory setpoint control, and the pathophysiology of the chronic hyperparathyroid state can be further investigated. The MEN1 tumor suppressor is the only other gene to date with an established role in the pathogenesis of sporadic parathyroid adenomatosis. Specific clonal alterations involving somatic mutation and/or deletion of both MEN1 alleles have been demonstrated in about 15-20% of sporadic parathyroid adenomas. Allelic losses on 11q occur in roughly twice this number of adenomas, raising the still-unresolved possibility that an additional tumor suppressor gene on 11q may be the functional target of many of these acquired deletions. A mouse model of MEN1 deficiency causes a phenotype that includes parathyroid hypercellularity albeit unaccompanied by biochemical hyperparathyroidism, and additional mouse models in which menin deficiency is targeted to the parathyroids will likely provide additional important insights. The MEN1 gene product menin may have a role in transcriptional regulation involving JunD; several other menin-interacting proteins have also been identified. The in vivo mechanism of menin's actions, with special attention to its role as a parathyroid oncosuppressor, will be important to establish, as will the potential interrelationships between these pathways and those involving cyclin D1. A number of genes, put forth as candidate tumor suppressors based on their genomic locations, roles in familial disease, and/or other relevant biological functions, have been examined for pathogenetic mutations in sporadic parathyroid tumors with negative results; these include the calcium-sensing receptor protein (CaR), vitamin-D receptor (VDR), and RET. However, the CaR, which when partially or markedly deficient because of germline mutation can cause familial hypocalciuric hypercalcemia or neonatal severe hyperparathyroidism, must still be considered as having a potentially important secondary role in the manifestations of sporadic parathyroid tumors. Future goals include identifying additional parathyroid oncogenes and tumor suppressor genes; exploiting tools of complex trait genetics to ascertain whether development of "sporadic" hyperparathyroidism might be influenced by predisposing polymorphic alleles in the population; obtaining molecular insights into the relationship between proliferative and hormone regulatory abnormalities of hyperparathyroidism; and obtaining molecular insights into the observed association of parathyroid neoplasia with exposure to ionizing irradiation and with the postmenopausal state.