ABSTRACT Introduction: Parathyroid tumors are clinically characterized by parathormone (PTH) hypersecretion from the parathyroid glands which become less sensitive to extracellular variations of calcium. This would seems caused by a decreased sensitivity to calcium by the calcium-sensing receptor (CASR), a receptor expressed on the apical membrane of parathyroid glands involved in the calcium homeostasis and able to sense changes of extracellular calcium ions. In fact, in these kind of tumours the CASR was down-regulated, but this does not seem to be due either to activating mutations of the CASR gene nor to a promoter hypermethylation of the gene. Therefore, our hypothesis was that it could be involved in this pathway another protein, Filamin A (FLNA), which is known to interact with the CASR at its C-terminal portion at level of 907-997 residues taking part in the stabilization of the receptor in the membrane. Just at level of these residues of CASR, it has been identified a non-conservative polymorphism Arg990Gly leading to a gain of function, an increased sensibility to calcimimetic R-568 and a different pathway than CASR wild-type. Aim: To investigate the role of FLNA in human parathyroid tumour tissues (adenomas and carcinomas) and to evaluate the interaction between FLNA and CASR, both wild-type and polymorphic, in absence or presence of calcimimetic R-568. Materials and Methods: The expression of FLNA and CASR was evaluated by immunofluorescence and immunohistochemistry in parathyroid adenomas and carcinomas, and by immunofluorescence in HEK-293 cells. CASR and FLNA mRNA levels were measured with Real-Time PCR in 74 parathyroid adenomas; parathyroid adenomas of the same samples were genotyped for the SNP R990G using the technique Taqman genotyping assay. FLNA was silenced with a Short interfering RNA in human embryonic kidney cells (HEK-293) transfected with wild-type or polymorphic CASR. The CASR activity was assessed by measuring the activity of ERK 1/2 by Western blot, both in absence or presence of calcimimetic R-568. Results: Immunohistochemistry of parathyroid adenomas and carcinomas showed a decrease of FLNA expression related to the degree of tumour malignancy. FLNA and CASR mRNA levels were variable in relation to adenoma tissue analysed, but its were positively correlated (r2=0.223 P<0.0001). The correlation between FLNA and CASR mRNA levels and the minor allele 990G did not shown significant differences among two groups (AG and GG), but it was observed a trend between the minor allele 990G and the higher expression of CASR than major allele 990R (wild-type). In agreement with these data, the genotype-phenotype correlation analysis showed serum PTH levels reduced in patients carriers of the minor. Western Blot data showed a decrease of expression and activity in both forms of the receptor (WT CASR and R990G CASR) in the absence of FLNA. The presence of calcimimetic R-568 seems to hide the differences in absence of FLNA, taking the gain of function of the CASR R990G. Conclusions: In human parathyroid tumours both FLNA and CASR were down-regulated and their mRNA levels were positive correlated. In particular, the trend observed between FLNA and CASR990G mRNA expression level could indicate a potential protective role of this SNP in parathyroid tumours, with a yet unknown mechanism. Furthermore, this study allowed us to show that FLNA is required for the activation of ERK 1/2 pathway activated both by CASR wild-type and polymorphic, even if in presence of R-568 the FLNA seems not to be necessary for the pathway activation.

RUOLO DELLA FILAMINA A NEI TUMORI PARATIROIDEI UMANI E IL SUO EFFETTO SULL'ATTIVITA' DEL CALCIUM SENSING RECEPTOR / C. Brasacchio ; tutore: L. Soldati. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2015 Dec 17. ((28. ciclo, Anno Accademico 2015.

RUOLO DELLA FILAMINA A NEI TUMORI PARATIROIDEI UMANI E IL SUO EFFETTO SULL'ATTIVITA' DEL CALCIUM SENSING RECEPTOR

C. Brasacchio
2015-12-17

Abstract

ABSTRACT Introduction: Parathyroid tumors are clinically characterized by parathormone (PTH) hypersecretion from the parathyroid glands which become less sensitive to extracellular variations of calcium. This would seems caused by a decreased sensitivity to calcium by the calcium-sensing receptor (CASR), a receptor expressed on the apical membrane of parathyroid glands involved in the calcium homeostasis and able to sense changes of extracellular calcium ions. In fact, in these kind of tumours the CASR was down-regulated, but this does not seem to be due either to activating mutations of the CASR gene nor to a promoter hypermethylation of the gene. Therefore, our hypothesis was that it could be involved in this pathway another protein, Filamin A (FLNA), which is known to interact with the CASR at its C-terminal portion at level of 907-997 residues taking part in the stabilization of the receptor in the membrane. Just at level of these residues of CASR, it has been identified a non-conservative polymorphism Arg990Gly leading to a gain of function, an increased sensibility to calcimimetic R-568 and a different pathway than CASR wild-type. Aim: To investigate the role of FLNA in human parathyroid tumour tissues (adenomas and carcinomas) and to evaluate the interaction between FLNA and CASR, both wild-type and polymorphic, in absence or presence of calcimimetic R-568. Materials and Methods: The expression of FLNA and CASR was evaluated by immunofluorescence and immunohistochemistry in parathyroid adenomas and carcinomas, and by immunofluorescence in HEK-293 cells. CASR and FLNA mRNA levels were measured with Real-Time PCR in 74 parathyroid adenomas; parathyroid adenomas of the same samples were genotyped for the SNP R990G using the technique Taqman genotyping assay. FLNA was silenced with a Short interfering RNA in human embryonic kidney cells (HEK-293) transfected with wild-type or polymorphic CASR. The CASR activity was assessed by measuring the activity of ERK 1/2 by Western blot, both in absence or presence of calcimimetic R-568. Results: Immunohistochemistry of parathyroid adenomas and carcinomas showed a decrease of FLNA expression related to the degree of tumour malignancy. FLNA and CASR mRNA levels were variable in relation to adenoma tissue analysed, but its were positively correlated (r2=0.223 P<0.0001). The correlation between FLNA and CASR mRNA levels and the minor allele 990G did not shown significant differences among two groups (AG and GG), but it was observed a trend between the minor allele 990G and the higher expression of CASR than major allele 990R (wild-type). In agreement with these data, the genotype-phenotype correlation analysis showed serum PTH levels reduced in patients carriers of the minor. Western Blot data showed a decrease of expression and activity in both forms of the receptor (WT CASR and R990G CASR) in the absence of FLNA. The presence of calcimimetic R-568 seems to hide the differences in absence of FLNA, taking the gain of function of the CASR R990G. Conclusions: In human parathyroid tumours both FLNA and CASR were down-regulated and their mRNA levels were positive correlated. In particular, the trend observed between FLNA and CASR990G mRNA expression level could indicate a potential protective role of this SNP in parathyroid tumours, with a yet unknown mechanism. Furthermore, this study allowed us to show that FLNA is required for the activation of ERK 1/2 pathway activated both by CASR wild-type and polymorphic, even if in presence of R-568 the FLNA seems not to be necessary for the pathway activation.
SOLDATI, LAURA
Settore MED/06 - Oncologia Medica
RUOLO DELLA FILAMINA A NEI TUMORI PARATIROIDEI UMANI E IL SUO EFFETTO SULL'ATTIVITA' DEL CALCIUM SENSING RECEPTOR / C. Brasacchio ; tutore: L. Soldati. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2015 Dec 17. ((28. ciclo, Anno Accademico 2015.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/348007
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