Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.

Genetic Deletion of Menin in Mouse Mesenchymal Stem Cells: An Experimental and Computational Analysis.

Loss-of-function mutations in the MEN1 tumor-suppressor gene cause the multiple endocrine neoplasia type 1 syndrome. Menin, the MEN1 gene product, is expressed in many tissues, including bone, where its function remains elusive. We conditionally inactivated menin in mesenchymal stem cells (MSCs) using paired-related homeobox 1 (Prx1)-Cre and compared resultant skeletal phenotypes of Prx1-Cre;Men1 f/f menin-knockout mice (KO) and wild-type controls using in vivo and in vitro experimental approaches and mechanics simulation. Dual-energy X-ray absorptiometry demonstrated significantly reduced bone mineral density, and 3-dimensional micro-CT imaging revealed a decrease in trabecular bone volume, altered trabecular structure, and an increase in trabecular separation in KO mice at 6 and 9 months of age. Numbers of osteoblasts were unaltered, and dynamic histomorphometry demonstrated unaltered bone formation; however, osteoclast number and activity and receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) mRNA profiles were increased, supporting increased osteoclastogenesis and bone resorption. In vitro, proliferative capabilities of bone marrow stem cells and differentiation of osteoblasts and mineralization were unaltered; however, osteoclast generation was increased. Gross femur geometrical alterations observed included significant reductions in length and in mid-metaphyseal cross-sectional area. Atomic force microscopy demonstrated significant decreases in elasticity of both cortical and trabecular bone at the nanoscale, whereas three-point bending tests demonstrated a 30% reduction in bone stiffness; finite element analysis showed morphological changes of the femur microgeometry and a significantly diminished femur flexural rigidity. The biomechanical results demonstrated the detrimental outcome of the accelerated osteoclastic bone resorption. Our studies have a twofold implication; first, MEN1 deletion from MSCs can negatively regulate bone mass and bone biomechanics, and second, the experimental and computational biomechanical analyses employed in the present study should be applicable for improved phenotypic characterization of murine bone. Furthermore, our findings of critical menin function in bone may underpin the more severe skeletal phenotype found in hyperparathyroidism associated with loss-of-function of the MEN1 gene. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Novel Glial Cells Missing-2 (GCM2) variants in parathyroid disorders.

OBJECTIVE: The aim of this study was to analyze variants of the gene glial cells missing-2 (GCM2), encoding a parathyroid cell-specific transcription factor, in familial hypoparathyroidism and in familial isolated hyperparathyroidism (FIHP) without and with parathyroid carcinoma. DESIGN: We characterized 2 families with hypoparathyroidism and 19 with FIHP in which we examined the mechanism of action of GCM2 variants. METHODS: Leukocyte DNA of hypoparathyroid individuals was Sanger sequenced for CASR, PTH, GNA11 and GCM2 mutations. DNA of hyperparathyroid individuals underwent MEN1, CDKN1B, CDC73, CASR, RET and GCM2 sequencing. The actions of identified GCM2 variants were evaluated by in vitro functional analyses. RESULTS: A novel homozygous p.R67C GCM2 mutation which failed to stimulate transcriptional activity in a luciferase assay was identified in affected members of two hypoparathyroid families. Oligonucleotide pull-down assay and in silico structural modeling indicated that this mutant had lost the ability to bind the consensus GCM recognition sequence of DNA. Two novel (p.I383M and p.T386S) and one previously reported (p.Y394S) heterozygous GCM2 variants that lie within a C-terminal conserved inhibitory domain were identified in three affected individuals of the hyperparathyroid families. One family member, heterozygous for p.I138M, had parathyroid carcinoma (PC), and a heterozygous p.V382M variant was found in another patient affected by sporadic PC. These variants exerted significantly enhanced in vitrotranscriptional activity, including increased stimulation of the PTH promoter. CONCLUSIONS: We provide evidence that two novel GCM2 R67C inactivating mutations with an inability to bind DNA are causative of hypoparathyroidism. Additionally, we provide evidence that two novel GCM2 variants increased transactivation of the PTH promoter in vitro and are associated with FIHP. Furthermore, our studies suggest that activating GCM2 variants may contribute to facilitating more aggressive parathyroid disease.

The three common polymorphisms p.A986S, p.R990G and p.Q1011E in the calcium sensing receptor (CASR) are not associated with chronic pancreatitis.

BACKGROUND: The calcium sensing receptor (CASR) is a G protein-coupled receptor that is responsible for assessing extracellular Ca2+ levels and thus plays a crucial role in calcium homeostasis. Hypercalcemia is a metabolic risk factor for pancreatitis and rare CASR variants have been described in patients with chronic pancreatitis. At the carboxy-terminal tail of CASR, there is a cluster of three common polymorphisms, p.A986S (rs1801725), p.R990G (rs1042636) and p.Q1011E (rs1801726), which have been associated with chronic pancreatitis in various studies, but with conflicting results. METHODS: We examined 542 German and 339 French patients with chronic pancreatitis as well as 1025 German controls for the 3 common CASR polymorphism by melting curve analysis. For comparison, we used genotype data from 583 French controls from a previous study. In addition, we functionally analyzed the three variants by NFAT and SRE luciferase reporter systems as well as Western blotting and verified cell surface expression by ELISA. RESULTS: In both cohorts, neither the genotype nor the allele frequencies differed significantly between patients and controls. In both luciferase assays, p.R990G showed a significant leftward shift, indicating an increased responsiveness of the receptor. p.A986S showed a leftward shift in the SRE but not in the NFAT reporter assay, while the responsiveness of p.Q1011E did not differ from the wild-type. These functional studies therefore do not support the contributions of variant CASR to increasing the risk of pancreatitis. CONCLUSIONS: The three frequent CASR polymorphisms are unlikely to increase the risk for chronic pancreatitis.

Homozygous Calcium-Sensing Receptor Polymorphism R544Q Presents as Hypocalcemic Hypoparathyroidism.

Context: Autosomal dominant hypocalcemia type 1 (ADH1) is caused by heterozygous activating mutations in the calcium-sensing receptor gene (CASR). Whether polymorphisms that are benign in the heterozygous state pathologically alter receptor function in the homozygous state is unknown. Objective: To identify the genetic defect in an adolescent female with a history of surgery for bilateral cataracts and seizures. The patient has hypocalcemia, hyperphosphatemia, and low serum PTH level. The parents of the proband are healthy. Methods: Mutation testing of PTH, GNA11, GCM2, and CASR was done on leukocyte DNA of the proband. Functional analysis in transfected cells was conducted on the gene variant identified. Public single nucleotide polymorphism (SNP) databases were searched for the presence of the variant allele. Results: No mutations were identified in PTH, GNA11, and GCM2 in the proband. However, a germline homozygous variant (c.1631G>A; p.R544Q) in exon 6 of the CASR was identified. Both parents are heterozygous for the variant. The variant allele frequency was near 0.1% in SNP databases. By in vitro functional analysis, the variant was significantly more potent in stimulating both the Ca2+i and MAPK signaling pathways than wild type when transfected alone (P < 0.05) but not when transfected together with wild type. The overactivity of the mutant CaSR is due to loss of a critical structural cation-π interaction. Conclusions: The patient's hypoparathyroidism is due to homozygosity of a variant in the CASR that normally has weak or no phenotypic expression in heterozygosity. Although rare, this has important implications for genetic counseling and clinical management.

Transforming Growth Factor-beta Regulation of Ephrin Type-A Receptor 4 Signaling in Breast Cancer Cellular Migration.

Breast cancer consists of a range of tumor subtypes with different clinical characteristics, disease prognosis, and treatment-response. Luminal breast cancer has the best prognosis while basal-like breast cancer (BLBC) represents the worst subtype. Transforming growth factor-beta (TGFß) plays a prominent role in stimulating the migration and invasion of malignant breast cancer cells contributing to tumor progression. In this study, we identified the Ephrin type-A receptor 4 (EPHA4) as a novel target of TGFß in breast cancer. Moreover, we show that TGFß induction of EPHA4 gene expression is specific to basal-like tumors and is required for TGFß-mediated cell migration. We further addressed the mechanism and found EPHA4 to be required for TGFß-mediated cell migration in breast cancer through TGFß-induced short term and long term activation of RhoGTPases. Finally, our data revealed a strong association between high EPHA4 expression and advanced tumor stage, aggressive BLBC molecular subtype and poor prognosis. Importantly, we found significant co-expression of EPHA4 and the TGFß receptor type-2 (TGFßR2) in breast cancer subtypes associated with increased tumor relapse and drug resistance. Together, this study highlight the important role of the TGFß/EPHA4 signaling axis in mediating tumor aggressiveness and poor patient survival in human breast cancer.

Calcium-Sensing Receptor Gene: Regulation of Expression.

The human calcium-sensing receptor gene (CASR) has 8 exons, and localizes to chromosome 3q. Exons 1A and 1B encode alternative 5'-untranslated regions (UTRs) that splice to exon 2 encoding the AUG initiation codon. Exons 2-7 encode the CaSR protein of 1078 amino acids. Promoter P1 has TATA and CCAAT boxes upstream of exon 1A, and promoter P2 has Sp1/3 motifs at the start site of exon 1B. Exon 1A transcripts from the P1 promoter are reduced in parathyroid tumors and colon carcinomas. Studies of colon carcinomas and neuroblastomas have emphasized the importance of epigenetic changes-promoter methylation of the GC-rich P2 promoter, histone acetylation-as well as involvement of microRNAs in bringing about CASR gene silencing and reduced CaSR expression. Functional cis-elements in the CASR promoters responsive to 1,25-dihydroxyvitamin D [1,25(OH)2D], proinflammatory cytokines, and the transcription factor glial cells missing-2 (GCM2) have been characterized. Reduced levels of CaSR and reduced responsiveness to active vitamin D in parathyroid neoplasia and colon carcinoma may blunt the "tumor suppressor" activity of the CaSR. The hypocalcemia of critically ill patients with burn injury or sepsis is associated with CASR gene upregulation by TNF-alpha and IL-1beta via kappaB elements, and by IL-6 via Stat1/3 and Sp1/3 elements in the CASR gene promoters, respectively. The CASR is transactivated by GCM2-the expression of which is essential for parathyroid gland development. Hyperactive forms of GCM2 may contribute to later parathyroid hyperactivity or tumorigenesis. The expression of the CaSR-the calciostat-is regulated physiologically and pathophysiologically at the gene level.

Calcium-sensing receptor, proinflammatory cytokines and calcium homeostasis.

The calcium-sensing receptor (CaSR) expressed in the parathyroid gland and the kidney tubule acts as the calciostat and orchestrates blood calcium homeostasis by modulating production and release of parathyroid hormone (PTH) and active vitamin D that influence Ca(2+) fluxes across the bone, kidney and intestine. Here we consider the role of the CaSR as a responder to proinflammatory cytokines released as part of the innate immune response to tissue injury and inflammation with resetting of the calciostat on the one hand and as a promoter and mediator of the initial inflammatory response on the other. The importance of the CaSR in systemic calcium homeostasis is exemplified by the fact that inactivating and activating mutations in the gene result in hypercalcemia and hypocalcemia, respectively. Proinflammatory cytokines interleukin-1ß and interleukin-6 upregulate CaSR expression in parathyroid and kidney and do this through defined response elements in the CASR gene promoters. This results in decreased serum PTH and 1,25-dihydroxyvitamin D and calcium levels. This is likely to underlie the hypocalcemia that commonly occurs in critically ill patients, those with burn injury and sepsis, for example. The level of calcium in extracellular fluid bathing necrotic cells is often elevated and acts as a chemokine to attract monocytes/macrophages that express the CaSR to sites of tissue injury. Elevated levels of calcium acting via the CaSR can function as a danger signal that stimulates assembly of myeloid cell cytosolic multiprotein inflammasomes resulting in maturation of the proinflammatory cytokine IL-1ß by caspase-1. Thus the CaSR is both promoter of and responder to the inflammation.

The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFß-induced Autophagy.

TGFß is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFß signaling and autophagy is context-dependent and remains unclear. TGFß-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFß in hepatocarcinoma cells. In the present study, we define a novel process of TGFß-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFß is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFß tumor suppressive effects. We further determined that TGFß induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFß induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFß signaling in tumor suppression.

Cinacalcet Treatment in an Adolescent With Concurrent 22q11.2 Deletion Syndrome and Familial Hypocalciuric Hypercalcemia Type 3 Caused by AP2S1 Mutation.

CONTEXT: The 22q11.2 deletion syndrome (DS) is a common multiple anomaly syndrome in which typical features include congenital heart defects, facial dysmorphism, and palatal anomalies. Hypocalcemia due to hypoparathyroidism is a common endocrine manifestation resulting from variable parathyroid hypoplasia, but hypercalcemia has not previously been reported in 22q11.2 DS. CASE DESCRIPTION: Our patient is a 16-year-old adolescent male with dysmorphic facial features and delayed motor and speech development. At 2 years of age, 22q11.2 DS was confirmed by fluorescence in situ hybridization. In contrast to hypoparathyroidism that is usually seen in 22q11.2 DS, this patient had early childhood-onset hypercalcemia with inappropriately high PTH levels and hypocalciuria. Genomic DNA was obtained from the proband and screened for calcium-sensing receptor (CASR) mutations with negative results. No parathyroid tissue could be localized by imaging or surgical exploration. As a result of symptomatic hypercalcemia, the patient was treated with a calcimimetic (cinacalcet). During the treatment, plasma calcium normalized with mild symptoms of hypocalcemia. After discontinuation of cinacalcet, calcium returned to high pretreatment levels. Further DNA analysis of adaptor protein-2 σ subunit (AP2S1) showed a heterozygous missense mutation c.44 G>T, resulting in a p.R15L substitution; the mutation was absent in the healthy parents and two siblings. CONCLUSIONS: Hypercalcemia in our patient with 22q11.2 DS could be explained by the de novo mutation in AP2S1. Identification of a genetic cause for hypercalcemia is helpful in guiding management and avoiding unnecessary treatment.

The leukemia inhibitory factor (LIF) and p21 mediate the TGFß tumor suppressive effects in human cutaneous melanoma.

BACKGROUND: Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFß) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion. METHODS: In this study, we aimed at elucidating the molecular mechanisms underlying TGFß-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays. RESULTS: We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFß in melanoma cells, defining LIF as a novel TGFß downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFß-mediated LIF expression is required for TGFß-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFß-mediated inhibition of cell migration. Moreover, we found that TGFß-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFß/LIF-mediated cell cycle arrest and TGFß-induced gene activation of several pro-apoptotic genes. CONCLUSIONS: Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFß to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.

Osteoblast menin regulates bone mass in vivo.

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-ß (TGF-ß) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-ß, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1(f/f)) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1(f/f) mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor ß/Smad signaling and associates with favorable breast cancer disease outcomes.

INTRODUCTION: This study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor ß (TGFß) pathway, which induces potent migration and invasion responses in breast cancer cells. METHODS: We surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFß/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFß-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging-based matrix degradation assay, respectively. RESULTS: Relatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFß through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFß/Smad signaling pathway. CONCLUSION: BCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFß/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3's molecular mechanism of action and highlight BCAR3 as a novel TGFß/Smad antagonist in breast cancer.

Increased prevalence of the GCM2 polymorphism, Y282D, in primary hyperparathyroidism: analysis of three Italian cohorts.

CONTEXT: Glial cells missing-2 (GCM2) is key for parathyroid gland organogenesis. Its persistent expression in the adult parathyroid raises the possibility that overactive forms play a role in the evolution of parathyroid hyperactivity or tumorigenesis. A GCM2 c.844T → G; p.Y282D missense variant has been described within a transactivation inhibitory domain (amino acids 263-352). OBJECTIVE: The aims of the study were to 1) assess the frequency of Y282D in Italian primary hyperparathyroidism (PHPT) and control (C) populations, 2) test for association of 282D with PHPT and its phenotypic features, and 3) compare the transactivation potency of GCM2 282D relative to wild-type Y282. SUBJECTS AND METHODS: Subjects included a large southern Italian cohort (310 PHPT and 433 C) and 2 replication cohorts from northern Italy. Association of 282D with PHPT was tested in all cohorts and with phenotypic features in the larger PHPT cohort. An in vitro GCM promoter-luciferase reporter assay was conducted in HEK293 cells. RESULTS: 282D was significantly increased in the PHPT group, with a minor allele frequency of 0.066 compared with 0.029 in the C group (P = .0008), in the discovery cohort and was more prevalent in the replication cohorts. Combined analysis (510 PHPT and 665 C) yielded a likelihood ratio of 2.27 (95% confidence interval = 1.50-3.42; P < .0001). The 282D variant was not associated with serum calcium, phosphate, creatinine, or PTH levels or with bone mineral density, fractures, or renal stones in the PHPT group. The 282D variant had significantly greater transcriptional activity than the wild-type Y282 (17× basal vs 12× basal; P < 0.05). CONCLUSION: The higher frequency of GCM2 282D in PHPT and enhanced transcriptional activity of this variant supports the notion that it could contribute causally to parathyroid tumorigenesis.

Novel homozygous inactivating mutation of the calcium-sensing receptor gene (CASR) in neonatal severe hyperparathyroidism-lack of effect of cinacalcet.

BACKGROUND: NSHPT is a life-threatening disorder caused by homozygous inactivating calcium-sensing receptor (CASR) mutations. In some cases, the CaSR allosteric activator, cinacalcet, may reduce serum PTH and calcium levels, but surgery is the treatment of choice. OBJECTIVE: To describe a case of NSHPT unresponsive to cinacalcet. PATIENT AND RESULTS: A 23-day-old girl was admitted with hypercalcemia, hypotonia, bell-shaped chest and respiratory distress. The parents were first-degree cousins once removed. Serum Ca was 4.75 mmol/l (N: 2.10-2.62), P: 0.83 mmol/l (1.55-2.64), PTH: 1096 pg/ml (9-52) and urinary Ca/Cr ratio: 0.5mg/mg. First, calcitonin was given (10 IU/kg × 4/day), and then 2 days later, pamidronate (0.5mg/kg) for 2 days. Doses of cinacalcet were given daily from day 28 of life starting at 30 mg/m2 and increasing to 90 mg/m2 on day 43. On day 33, 6 days after pamidronate, serum Ca levels had fallen to 2.5 mmol/l but, thereafter, rose to 5 mmol/l despite the cinacalcet. Total parathyroidectomy was performed at day 45. Hungry bone disease after surgery required daily Ca replacement and calcitriol for 18 days. At 3 months, the girl was mildly hypercalcemic, with no supplementation, and at 6 months, she developed hypocalcemia and has since been maintained on Ca and calcitriol. By CASR mutation analysis, the infant was homozygous and both parents heterozygous for a deletion-frameshift mutation. CONCLUSION: The predicted nonfunctional CaSR is consistent with lack of response to cinacalcet, but total parathyroidectomy was successful. An empiric trial of the drug and/or prompt mutation testing should help minimize the period of unnecessary pharmacotherapy.

Codon Arg15 mutations of the AP2S1 gene: common occurrence in familial hypocalciuric hypercalcemia cases negative for calcium-sensing receptor (CASR) mutations.

CONTEXT: Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant disorder with three known subtypes: FHH1, FHH2, and FHH3. About 65% of FHH cases are FHH1, caused by inactivating mutations of the calcium-sensing receptor (CASR) gene. FHH3 was recently found to be caused by codon Arg15 (p.R15) mutations in the adaptor-related protein complex 2, σ-2 subunit that interacts with the CaSR and is encoded by the AP2S1 gene. OBJECTIVE: The objective of the study was to assess the prevalence of AP2S1 mutations, and describe the phenotype of FHH3, in an independent cohort of FHH subjects lacking CASR mutations. PATIENTS AND METHODS: Thirty-nine patients presenting with some combination of hypercalcemia, hypermagnesemia, nonsuppressed serum PTH levels, and reduced urinary calcium excretion were studied. Exon 2 of the AP2S1 gene was PCR amplified from patient genomic DNA and Sanger sequenced. The presence of p.R15 mutations was confirmed by restriction enzyme analysis. RESULTS: Five of the 39 subjects had AP2S1 p.R15 mutations, a frequency of 13%. The three recurrent mutations reported previously were all found in our cohort (p.R15C in two, p.R15L in two, and p.R15H in one subject). The FHH3 phenotype did not differ materially from that of FHH1 due to CASR mutations. CONCLUSIONS: The results affirm that a significant number of patients suspected of having FHH but proven negative for CASR mutation have AP2S1 p.R15 mutations. Screening for AP2S1 p.R15 mutations in such cases should be considered, given the clinical benefits (avoiding unnecessary parathyroidectomy) that have already been demonstrated for CASR screening in FHH1.

Involvement of the osteoinductive factors, Tmem119 and BMP-2, and the ER stress response PERK-eIF2α-ATF4 pathway in the commitment of myoblastic into osteoblastic cells.

The osteoinductive factors BMP-2 and Tmem119 that promote the differentiation of myoblasts into osteoblasts, each increase the levels of the other. However, the relative contributions of BMP-2 and Tmem119 to the osteogenic differentiation and the mechanisms involved are incompletely understood. In the present study, we examined the relationship among BMP-2, Tmem119, and the PERK-eIF2α-ATF4 endoplasmic reticulum (ER) stress response pathway in the differentiation of C2C12 myoblasts into osteoblastic cells. Both BMP-2 and Tmem119 induced levels of the osteoblast markers Runx2, Osterix, Col1a1, ALP, and osteocalcin, as well as mineralization. BMP-2 activation of the ER stress sensor PERK stimulated phosphorylation of eIF2α and led to increased biosynthesis of the osteoblast differentiation factor ATF4. When dephosphorylation of eIF2α was blocked by the selective inhibitor salubrinal, the osteogenic effects of BMP-2 and Tmem119 were enhanced further. Although BMP-2 stimulated both P-eIF2α and ATF4 levels, Tmem119 had no effect on P-eIF2α but stimulated ATF4 only. Reduction in endogenous Tmem119 levels by siRNA reduced both basal and BMP-2-stimulated levels of the ATF4 protein. In conclusion, BMP-2 stimulates differentiation of myoblasts into osteoblasts via the PERK-eIF2α-ATF4 pathway but in addition stimulates Tmem119, which itself increases ATF4. Hence, BMP-2 stimulates ATF4 both dependently and independently of the PERK-eIF2α ER stress response pathway.

Identification and functional characterization of three NoLS (nucleolar localisation signals) mutations of the CDC73 gene.

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.

The CASR gene: alternative splicing and transcriptional control, and calcium-sensing receptor (CaSR) protein: structure and ligand binding sites.

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor encoded by a single copy gene. The human CASR gene spans ~103-kb and has eight exons. Promoters P1 and P2 drive transcription of exons 1A and 1B, respectively, encoding alternative 5'-UTRs that splice to exon 2 encoding the common part of the 5'-UTR. Exons 2-7 encode the CaSR protein of 1078 amino acids. Functional elements responsive to 1,25-dihydroxyvitamin D, proinflammatory cytokines, and glial cells missing-2 are present in the CASR promoters. Evolutionarily, the exon structure, first seen in aquatic vertebrates, is well-conserved with a single linkage disequilibrium haplotype block for protein coding exons 2-7. Structural features of the human CaSR protein are: an N-terminal signal peptide (19 amino acids (aa)); an extracellular domain (~600 aa) having a bi-lobed Venus Flytrap (VFT) domain with several Ca(2+)-binding sites; and a nine-cysteines domain that transduces the activation signal to the 7-transmembrane domain (250 aa) and the C-terminal tail (216 aa).