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Molecular mechanisms involved in the protective effect of 17β-estradiol and testosterone against apoptosis in skeletal muscle cells
Abstracts There is a global trend to use natural bioactive compounds such as phytoestrogens, presents in a wide variety of foods. They possess numerous health benefits including anticancer, anti-oxidant and cardiovascular effects. Genistein (GEN) and Quercetin (QUE) are phytoestrogens with great structural similarity to human estrogen. We evaluated the effects of GEN and QUE on estrogen receptor-positive cells: MC3T3 murine preosteoblasts and MCF-7 human breast cancer cell line. Cell migration is important in cicatrization, implants, fracture repair, immune response, among others. Errors during this process have pathological consequences, including tumor formation and metastasis. The wound healing assay show that GEN (0.01, 1 μM) and QUE (1 μM, 10 μM) stimulate the closure and wound healing after 24 h of preosteoblasts treatment; while QUE decreases tumoral cell migration. Trypan blue assay indicates that after 48 h treatment, both GEN and QUE (1 μM) significantly increase proliferation of preosteoblasts in 145.66 ± 5.76% and 150.16 ± 5.58% (p ˂ 0.05), respectively, not affecting tumoral number of cells. 100 μM QUE inhibits cell proliferation in both cell lines, with a major effect on tumoral cells. When antioxidant activity was evaluated on preoteoblasts, we found that pre-treatment of cells for 48 h with 1 μM GEN was more effective than QUE in the prevention of H2O2-induced cell (Ctrl. vs. GEN: 26.8 ± 4.21 vs. 7.6 ± 1.8, p ˂ 0.05). Phosphorilation levels of Akt and Bad evaluated by Western blot indicate that 100 μM QUE decreases preosteoblastic pAkt, leading to a decrease in phosphorilation levels of the proapoptotic protein Bad. Altogether, polyphenolrich diets may be a good alternative therapy for bone and tumoral pathologies, because of its opposites but beneficial effects in both events.
doi:10.1016/j.bone.2015.12.046
Prevalence of secondary causes in a population of premenopausal women with low bone mass V.S. Farias, M.B. Zanchetta Instituto de Diagnóstico e Investigaciones Metabólicas (IDIM), Argentina E-mail address: [email protected] (V.S. Farias) Low bone mass in premenopausal women is a relatively frequent consultation and the prevalence of secondary causes is variable in the literature and has been described between 50–90%. Objective: To evaluate the prevalence of secondary causes of low bone mass in a group of premenopausal women. Material and methods: A retrospective, descriptive and observational study, analyzing the electronic medical records of all women under 50 years who consulted for Low Bone Mass between 01/06/13 and 01/29/15. Premenopausal low bone mass was defined for ISCD (The International Society for Clinical Densitometry): patients with Z-score less than or equal to − 2 in lumbar spine or hip. Preliminary results: The mean age was 38 years (range 20–50 years) and the mean values of DXA were: lumbar spine: 0.912 g/cm2, total hip: 0.754 g/cm2 and femoral neck: 0.762 g/cm2. Of the 50 women tested, 64% had considered a secondary cause. Only 22% of women had a single cause, the rest had multiple causes; and 36% (18 patients) had idiopathic bone mass. As for treatment, only 6 patients received treatment osteoactive, the remaining patients received only calcium, vitamin D and causes secondary correction. Conclusions: The 64% of the evaluated premenopausal women had at least a secondary cause of low bone mass and 12% had a history of fragility fractures. The main risk factors found were steroid use, hypoestrogenism, low weight and hypercalciuria. We consider it essential to carefully evaluate premenopausal women with low bone mass to rule out secondary causes and to take the necessary preventive measures to improve bone quality by reducing the future risk of osteoporotic fractures.
doi:10.1016/j.bone.2015.12.047
Rhabdomyosarcoma cells response to 1,25(OH)2-vitamin D3 inducing MAPK activation and VDR expression A.P. Irazoqui, R.L. Boland1, C.G. Buitrago INBIOSUR (UNS-CONICET) Bahía Blanca, Argentina 1 (in memory). E-mail address: [email protected] (C.G. Buitrago) Our laboratory has studied for many years the role of the steroid hormone 1α,25(OH)2-vitamin D3 [1,25D] in avian and mammalian skeletal muscle cells focused principally on vitamin D receptor (VDR) involvement and signal transduction pathways triggered by this hormone. Recently, we reported VDR and p38 MAPK participation in cell proliferation and differentiation induced by the hormone in the normal murine skeletal muscle cell line C2C12. There is no information about 1,25D actions in rhabdomyosarcoma (RMS) cells. RMS is a soft tissue cancer that affects skeletal muscle predominantly in child and elderly people and includes heterogeneous tumors of mesenchymal derivation which are genetically committed to the myogenic lineage, failing to complete terminal differentiation. In this work, using RD
71
cells, an embryonal rhabdomyosarcoma cell line, we evidenced that hormone promotes rapid effects. Our data indicate that 1 nM of 1,25D activates ERK1/2, p38 MAPK and the p38 MAPK upstream immediately kinases (MKK3/6) rapidly, evidencing that the hormone regulates non genomic transduction pathways in these cells. Moreover, the non receptor tyrosine kinase Src was also activated by 1,25D in RD cells. When ERK1/2 was inhibited with PD98059, Src activation was abolished. MKK3/6 activation was also dependent on ERK1/2 activation. Of relevance, 1,25D increases VDR expression in these cells and this event was dependent on p38 MAPK activation. The results indicate that 1,25D exerts rapid actions in rhabdomyosarcoma cells and open doors to investigate in deep hormone modulation of different signaling pathways in these cancer cells.
doi:10.1016/j.bone.2015.12.048
Molecular mechanisms involved in the protective effect of 17β-estradiol and testosterone against apoptosis in skeletal muscle cells L. Pronsato, A.B. La Colla, A.A. Vasconsuelo, L.M. Milanesi INBIOSUR, Universidad Nacional del Sur, Bahía Blanca, Argentina E-mail address: [email protected] (L.M. Milanesi) The loss of muscle mass and strength is a prevalent condition among the elderly, known as sarcopenia. Although the molecular mechanisms underlying this condition are not fully clarified, accumulating evidence suggests that apoptosis could be responsible for the loss of myocytes in adulthood. Of relevance to our work, sarcopenia has been associated to sex hormone deficit, observed in old adults. Previously we have demonstrated the protective effect, at morphological, physiological, biochemical and molecular level, of both 17β-estradiol (E2) and Testosterone (T) against H2O2-induced apoptosis in C2C12 skeletal muscle cell line. It has been established that the exposure of these cells to H2O2 represents a comparable phenotype to aged skeletal muscle, constituting a useful tool for the study of sarcopenia. The aim of our research is to deepen the understanding of the molecular mechanisms involved in the antiapoptotic action of both hormones. We have observed that H2O2 treatment induces the mitochondrial permeability transition pore (mPTP) opening, p53 activation and the increase of RNAm p66Shc, phosphorylation and mitochondrial protein translocation, where it exerts its apoptotic action. We observed that H2O2 induces JNK, PKD, PKCθ, PKCδ and PKCBI activation/phosphorylation, but not the PKCα/β2 one, acting some of these activated proteins, upstream of p66Shc apoptotic pathway. T or E2 treatment prior H2O2, reduces the mPTP opening and the levels of expression and/or phosphorylation of the proteins mentioned before. These results are in agreement with the observations indicating a protective effect of these steroids, and throw light on the knowledge of molecular mechanisms activated by each hormone to develop specific hormonal therapies.
doi:10.1016/j.bone.2015.12.049
Estimation of bone mineral content from routine abdominal CT studies: Correlation with densitometric data obtained by DXA H. Claus Hermberg, S. De Luca, F. Troncoso, M.P. Lozano, M. Rey, S. Boffa, M.J. Pozzo Hospital Alemán Buenos Aires, Endocrinology Service, Radiology Service Densitometry Division, Argentina E-mail address: [email protected] (M. Rey) Quantitative computed tomography (QCT) can exactly assess vertebral bone mineral content (BMC) using phantoms for calibration. Abdominal TC assesses in Hounsfield Units (HU) the average density of a region of interest (ROI) that includes the vertebral body. This value multiplied by area gives a magnitude that is proportional to BMC. With DXA, BMC of a vertebra projected in a ROI area of 10 mm thick centered at the midvertebral level represents that magnitude in CT axial section of the same vertebra. Nevertheless it is not equivalent, because DXA includes vertebral posterior elements. Taking into consideration theoretical similarities and differences between both techniques, the purpose of this study was to quantify the correlation between them using the basic applications of TC. Twenty eight adults who underwent abdominal TC for different reasons and DXA measurements within 6 month were included. A simple manual ROI of 10 mm thick was applied to L3. This ROI was manually delineated excluding vertebral pedicles. In thirteen patients an automatic ROI attenuation cut-off ≥70 UH was applied. We measured: density (UH), area (cm2) and maximum transverse diameter, density was multiplied by area. We correlate all parameters obtained by TC with the corresponding to DXA. Pearson correlations were used for analysis. We found significant correlation between DXA
doi:10.1016/j.bone.2015.12.046
Prevalence of secondary causes in a population of premenopausal women with low bone mass V.S. Farias, M.B. Zanchetta Instituto de Diagnóstico e Investigaciones Metabólicas (IDIM), Argentina E-mail address: [email protected] (V.S. Farias) Low bone mass in premenopausal women is a relatively frequent consultation and the prevalence of secondary causes is variable in the literature and has been described between 50–90%. Objective: To evaluate the prevalence of secondary causes of low bone mass in a group of premenopausal women. Material and methods: A retrospective, descriptive and observational study, analyzing the electronic medical records of all women under 50 years who consulted for Low Bone Mass between 01/06/13 and 01/29/15. Premenopausal low bone mass was defined for ISCD (The International Society for Clinical Densitometry): patients with Z-score less than or equal to − 2 in lumbar spine or hip. Preliminary results: The mean age was 38 years (range 20–50 years) and the mean values of DXA were: lumbar spine: 0.912 g/cm2, total hip: 0.754 g/cm2 and femoral neck: 0.762 g/cm2. Of the 50 women tested, 64% had considered a secondary cause. Only 22% of women had a single cause, the rest had multiple causes; and 36% (18 patients) had idiopathic bone mass. As for treatment, only 6 patients received treatment osteoactive, the remaining patients received only calcium, vitamin D and causes secondary correction. Conclusions: The 64% of the evaluated premenopausal women had at least a secondary cause of low bone mass and 12% had a history of fragility fractures. The main risk factors found were steroid use, hypoestrogenism, low weight and hypercalciuria. We consider it essential to carefully evaluate premenopausal women with low bone mass to rule out secondary causes and to take the necessary preventive measures to improve bone quality by reducing the future risk of osteoporotic fractures.
doi:10.1016/j.bone.2015.12.047
Rhabdomyosarcoma cells response to 1,25(OH)2-vitamin D3 inducing MAPK activation and VDR expression A.P. Irazoqui, R.L. Boland1, C.G. Buitrago INBIOSUR (UNS-CONICET) Bahía Blanca, Argentina 1 (in memory). E-mail address: [email protected] (C.G. Buitrago) Our laboratory has studied for many years the role of the steroid hormone 1α,25(OH)2-vitamin D3 [1,25D] in avian and mammalian skeletal muscle cells focused principally on vitamin D receptor (VDR) involvement and signal transduction pathways triggered by this hormone. Recently, we reported VDR and p38 MAPK participation in cell proliferation and differentiation induced by the hormone in the normal murine skeletal muscle cell line C2C12. There is no information about 1,25D actions in rhabdomyosarcoma (RMS) cells. RMS is a soft tissue cancer that affects skeletal muscle predominantly in child and elderly people and includes heterogeneous tumors of mesenchymal derivation which are genetically committed to the myogenic lineage, failing to complete terminal differentiation. In this work, using RD
71
cells, an embryonal rhabdomyosarcoma cell line, we evidenced that hormone promotes rapid effects. Our data indicate that 1 nM of 1,25D activates ERK1/2, p38 MAPK and the p38 MAPK upstream immediately kinases (MKK3/6) rapidly, evidencing that the hormone regulates non genomic transduction pathways in these cells. Moreover, the non receptor tyrosine kinase Src was also activated by 1,25D in RD cells. When ERK1/2 was inhibited with PD98059, Src activation was abolished. MKK3/6 activation was also dependent on ERK1/2 activation. Of relevance, 1,25D increases VDR expression in these cells and this event was dependent on p38 MAPK activation. The results indicate that 1,25D exerts rapid actions in rhabdomyosarcoma cells and open doors to investigate in deep hormone modulation of different signaling pathways in these cancer cells.
doi:10.1016/j.bone.2015.12.048
Molecular mechanisms involved in the protective effect of 17β-estradiol and testosterone against apoptosis in skeletal muscle cells L. Pronsato, A.B. La Colla, A.A. Vasconsuelo, L.M. Milanesi INBIOSUR, Universidad Nacional del Sur, Bahía Blanca, Argentina E-mail address: [email protected] (L.M. Milanesi) The loss of muscle mass and strength is a prevalent condition among the elderly, known as sarcopenia. Although the molecular mechanisms underlying this condition are not fully clarified, accumulating evidence suggests that apoptosis could be responsible for the loss of myocytes in adulthood. Of relevance to our work, sarcopenia has been associated to sex hormone deficit, observed in old adults. Previously we have demonstrated the protective effect, at morphological, physiological, biochemical and molecular level, of both 17β-estradiol (E2) and Testosterone (T) against H2O2-induced apoptosis in C2C12 skeletal muscle cell line. It has been established that the exposure of these cells to H2O2 represents a comparable phenotype to aged skeletal muscle, constituting a useful tool for the study of sarcopenia. The aim of our research is to deepen the understanding of the molecular mechanisms involved in the antiapoptotic action of both hormones. We have observed that H2O2 treatment induces the mitochondrial permeability transition pore (mPTP) opening, p53 activation and the increase of RNAm p66Shc, phosphorylation and mitochondrial protein translocation, where it exerts its apoptotic action. We observed that H2O2 induces JNK, PKD, PKCθ, PKCδ and PKCBI activation/phosphorylation, but not the PKCα/β2 one, acting some of these activated proteins, upstream of p66Shc apoptotic pathway. T or E2 treatment prior H2O2, reduces the mPTP opening and the levels of expression and/or phosphorylation of the proteins mentioned before. These results are in agreement with the observations indicating a protective effect of these steroids, and throw light on the knowledge of molecular mechanisms activated by each hormone to develop specific hormonal therapies.
doi:10.1016/j.bone.2015.12.049
Estimation of bone mineral content from routine abdominal CT studies: Correlation with densitometric data obtained by DXA H. Claus Hermberg, S. De Luca, F. Troncoso, M.P. Lozano, M. Rey, S. Boffa, M.J. Pozzo Hospital Alemán Buenos Aires, Endocrinology Service, Radiology Service Densitometry Division, Argentina E-mail address: [email protected] (M. Rey) Quantitative computed tomography (QCT) can exactly assess vertebral bone mineral content (BMC) using phantoms for calibration. Abdominal TC assesses in Hounsfield Units (HU) the average density of a region of interest (ROI) that includes the vertebral body. This value multiplied by area gives a magnitude that is proportional to BMC. With DXA, BMC of a vertebra projected in a ROI area of 10 mm thick centered at the midvertebral level represents that magnitude in CT axial section of the same vertebra. Nevertheless it is not equivalent, because DXA includes vertebral posterior elements. Taking into consideration theoretical similarities and differences between both techniques, the purpose of this study was to quantify the correlation between them using the basic applications of TC. Twenty eight adults who underwent abdominal TC for different reasons and DXA measurements within 6 month were included. A simple manual ROI of 10 mm thick was applied to L3. This ROI was manually delineated excluding vertebral pedicles. In thirteen patients an automatic ROI attenuation cut-off ≥70 UH was applied. We measured: density (UH), area (cm2) and maximum transverse diameter, density was multiplied by area. We correlate all parameters obtained by TC with the corresponding to DXA. Pearson correlations were used for analysis. We found significant correlation between DXA