Estrogen enhances human osteoblast survival and function via promotion of autophagy.

Estrogen increases bone formation by promoting mineralization and prolonging the lifespan of osteoblasts. To understand the underlying molecular mechanism/s, we identified estrogen-regulated proteins at different stages of human osteoblast differentiation using differential proteomics approach. Among the identified proteins, we observed that estrogen upregulated RAB3GAP1 on day 1 and 5 of differentiation. RAB3GAP1 is critically involved in the process of autophagy, a eukaryotic degradative pathway essential for cell survival. We, therefore, investigated the effect of estrogen on autophagy in differentiating human osteoblasts and their precursors, the mesenchymal stem cells (MSCs). MSCs exhibited high autophagic flux which declined during osteoblast differentiation, resulting in high basal apoptosis in osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing towards their longer lifespan. Further, MSCs were resistant against starvation-induced apoptosis, whereas, differentiating osteoblasts showed significant susceptibility towards it. Estrogen, in addition to promoting mineralization, protected differentiating osteoblasts from starvation-induced apoptosis by increasing autophagic flux. Autophagic flux in RAB3GAP1 knockdown osteoblasts appeared diminished, and showed increased apoptosis even in nutrient-rich conditions, and exhibited significantly impaired mineralization. However, irrespective of the presence of estrogen, starvation further enhanced apoptosis in these cells. Furthermore, estrogen failed to promote mineralization in these osteoblasts. Our study illustrates that autophagy is essential for human osteoblast survival and mineralization, and osteoblasts are susceptible to apoptosis due to reduced autophagy during differentiation. Estrogen, via upregulation of RAB3GAP1, promotes autophagy in osteoblasts during differentiation thereby increasing their survival and mineralization capacity. Our study demonstrates the positive role of autophagy in bone homeostasis.

Estrogen receptor α and ß gene polymorphism in relation to bone mineral density and lipid profile in Northeast Indian women.

Estrogen regulates bone homeostasis and has a cardio-protective effect. Its physiological functions are mediated through receptors (ER) whose expression can be regulated by presence or absence of polymorphisms. However, the association between ER polymorphisms and BMD as well as lipids are inconsistent. The aim of the study was to investigate whether polymorphisms in ESR are associated with bone mineral density (BMD) and lipids in a cohort of Indian women. We studied PvuII, XbaI polymorphisms in ESR1 and AluI, RsaI polymorphisms in ESR2 genes and their association with bone mineral density (BMD) and lipids in premenopausal (n = 293, mean age: 33.01 ±â€¯5.23 years) and postmenopausal (n = 145, mean age: 56.91 ±â€¯7.1 years) women from Northeast India. AluI and RsaI polymorphisms in ESR2 gene were associated with BMD in postmenopausal women. Logistic regression analysis adjusted for age, BMI, tobacco and alcohol consumption revealed that xx genotype in XbaI polymorphism is associated with osteopenia at spine (OR = 3.3, 95% CI = 1.067-10.204) in postmenopausal women suggesting that allele X is protective (OR = 0.419, 95% CI = 0.177-0.991). Genotype aa in AluI polymorphism, seemed to be protective (OR = 0.092 for osteopenia; OR = 0.152 for osteoporosis) at spine whereas A allele was associated with osteopenia at femur (OR = 2.123, 95% CI = 1.079-4.166) in postmenopausal women. Allele r of RsaI polymorphism, was associated with osteoporosis at spine (OR = 3.222, 95% CI = 1.302-7.96). Thus, AIuI polymorphism of ESR2 gene was associated with spinal and femoral BMD whereas RsaI only with spinal BMD in postmenopausal women and ESR genotypes were not associated with lipids.

LYN, a key mediator in estrogen-dependent suppression of osteoclast differentiation, survival, and function.

Estrogen insufficiency at menopause cause accelerated bone loss due to unwarranted differentiation and function of osteoclasts. Unraveling the underlying mechanism/s may identify mediators of estrogen action which can be targeted for improved management of osteoporosis. Towards this, we analyzed the effect of 17ß-estradiol on the proteomes of differentiating human osteoclasts. The major proteomic changes observed included upregulation of LYN by estrogen. We, therefore, investigated the effect of estrogen on osteoclast differentiation, survival, and function in control and LYN knockdown conditions. In control condition, estrogen treatment increased the apoptosis rate and suppressed the calcium signaling by reducing the intracellular Ca2+ levels as well as expression and activation of NFATc1 and c-Src during differentiation, resulting in reduced osteoclastogenesis. These osteoclasts were smaller in size with reduced extent of multinuclearity and produced significantly low levels of bone resorbing enzymes. They also exhibited disrupted sealing zone formation with low podosome density, impaired cell polarization and reduced resorption of dentine slices. Interestingly, in LYN knockdown condition, estrogen failed to induce apoptosis and inhibit activation of NFATc1 and c-Src. Compared to effect of estrogen on osteoclast in control condition, LYN knockdown osteoclasts did not show reduction in production of bone resorbing enzymes and had defined sealing zone formation with high podosome density with no impairment in cell polarization. They resorbed significant area on dentine slices. Thus, the inhibitory action of estrogen on osteoclast was severely restrained in LYN knockdown condition, demonstrating the importance of LYN as a key mediator of the effect of estrogen on osteoclastogenesis.

Association of high-density lipoprotein, triglycerides, and homocysteine with bone mineral density in young Indian tribal women.

This study investigated association between lipids and homocysteine (Hcy) with bone mineral density (BMD) in young women as opposed to previous studies on elderly women. HDL, triglyceride, and Hcy are significantly associated with BMD in young women and tobacco and alcohol consumption have no effect on this association. PURPOSE: The present study investigates whether the association of serum lipids and homocysteine (Hcy) with bone mineral density (BMD) reported mostly in elderly population can be generalized to young or premenopausal women, consequently suggesting screening of young women with low BMD for dyslipidemia or any cardiovascular events and vice versa. METHODS: Women (n = 293, aged 20-47 years) from Northeast India belonging to Tibeto-Burman origin were enrolled. Information about their physical and clinical attributes were collected by a structured questionnaire. Their BMDs at lumbar spine and femur were measured by dual-energy X-ray absorptiometry (DXA) and sera were profiled for lipid parameters and Hcy by auto-analyzer and ELISA, respectively. Women consuming tobacco and/or alcohol were grouped as consumers and others as non-consumers for the analysis. RESULTS: Positive correlation of BMD with HDL (spine and femur r = 0.38, p < 0.0001) and triglyceride (spine r = 0.534, p < 0.0001; femur r = 0.423, p < 0.0001) was observed, whereas Hcy correlated negatively with BMD (spine r = - 0.189, p = 0.0026; femur r = - 0.273, p < 0.0001). LDL showed a weak negative correlation with BMD (spine r = - 0.128, p = 0.0283; femur r = - 0.199, p = 0.0006). However, after adjusting for age, BMI, and consumption, HDL, triglyceride, and Hcy continued to show significant correlation with BMD at both the sites. Logistic regression analyses indicated that HDL, triglyceride, and Hcy were significant predictors of osteopenia and osteoporosis in our study cohort; however, consumption did not contribute to its prediction. CONCLUSION: Low levels of HDL and triglyceride and high levels of Hcy are significantly associated with osteopenia and osteoporosis in young Northeast Indian women.

Serum levels of phosphorylated heat shock protein 27 (pHSP27) are associated with bone mineral density in pre- & postmenopausal women: A pilot study.

BACKGROUND & OBJECTIVES: Phosphorylated heat shock protein 27 (pHSP27) has been implicated in the pathogenesis of osteoporosis. oxidative stress and proinflammatory cytokines, which are known to be involved in aetiology of osteoporosis, can trigger HSP27 phosphorylation. Since pHSP27 is present in circulation, it was hypothesized that serum pHSP27 would be elevated in low bone mineral density (BMD) condition and might serve as an indicator of osteoporosis/osteopenia. Hence, the aim of this study was to examine serum levels of pHSP27 in relation with BMD in pre- and postmenopausal women. METHODS: Premenopausal (30 to 40 yr) and postmenopausal (50 to 60 yr) women having either low BMD (osteopenia/osteoporosis) or high BMD were selected (n=80) from a prospective cohort (n=200). Serum levels of pHSP27; along with levels of oestradiol, malondialdehyde, total antioxidant capacity, interleukin (IL)-1, IL-6, tumour necrosis factor - alpha, (TNF-α), c-telopeptide fragments of collagen type I (CTX-1) and osteocalcin were estimated. RESULTS: The serum levels of pHSP27 were significantly elevated in low BMD groups in premenopausal and postmenopausal categories (p<0.05). It also exhibited a significant odds ratio (OR) to differentiate between low and high BMD in both premenopausal (OR=1.734, p=0.013) and postmenopausal (OR=1.463, p=0.042) categories. Additionally, area under the curve to predict low BMD was non-significantly higher for pHSP27 than CTX-1 in premenopausal and postmenopausal categories. INTERPRETATION & CONCLUSIONS: This study highlights a novel relation between serum pHSP27 and BMD in Indian women however, these findings need to be confirmed in larger studies.

Monocyte Proteomics Reveals Involvement of Phosphorylated HSP27 in the Pathogenesis of Osteoporosis.

Peripheral monocytes, precursors of osteoclasts, have emerged as important candidates for identifying proteins relevant to osteoporosis, a condition characterized by low Bone Mineral Density (BMD) and increased susceptibility for fractures. We employed 4-plex iTRAQ (isobaric tags for relative and absolute quantification) coupled with LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) to identify differentially expressed monocyte proteins from premenopausal and postmenopausal women with low versus high BMD. Of 1801 proteins identified, 45 were differentially abundant in low versus high BMD, with heat shock protein 27 (HSP27) distinctly upregulated in low BMD condition in both premenopausal and postmenopausal categories. Validation in individual samples (n = 80) using intracellular ELISA confirmed that total HSP27 (tHSP27) as well as phosphorylated HSP27 (pHSP27) was elevated in low BMD condition in both categories (P < 0.05). Further, using transwell assays, pHSP27, when placed in the upper chamber, could increase monocyte migration (P < 0.0001) and this was additive in combination with RANKL (receptor activator of NFkB ligand) placed in the lower chamber (P = 0.05). Effect of pHSP27 in monocyte migration towards bone milieu can result in increased osteoclast formation and thus contribute to pathogenesis of osteoporosis. Overall, this study reveals for the first time a novel link between monocyte HSP27 and BMD.

Age-related changes in bone turnover markers and ovarian hormones in premenopausal and postmenopausal Indian women.

This study characterizes age-related changes in bone turnover markers in relation to ovarian hormones. The data (N = 236) were divided into 5-year age bands and three groups: premenopausal (Group I, N = 139), perimenopausal (Group II, N = 30), and postmenopausal (Group III, N = 67). Age-related increases in mean parathyroid hormone (PTH), osteocalcin (OC), and collagen telopeptide (CTx) levels were observed. Women in Group II (N = 37) with osteopenia had lower levels of E1G (P<0.001) with normal FSH levels as compared to 50 women in the same group with normal bone mineral density (BMD). Their mean OC levels were reduced (P<0.05) and CTx levels were significantly elevated (P<0.01). The mean E1G levels were significantly lower (P<0.001) and mean CTx levels were significantly higher (P<0.001) in 30 perimenopausal women (Group II) compared to premenopausal women. In 28 postmenopausal women (group III) the mean BMD levels and E1G were significantly lower (P<0.001) with elevated FSH levels (P<0.001). Increased CTx levels (P<0.0001) reflected a higher rate of bone resorption. These observations suggest that perimenopausal women with low E1G, elevated FSH should be screened for osteoporosis, and it may be valid to combine simultaneous measurements of bone turnover markers with ovarian hormones when screening women at risk for osteoporosis.

Association of estrogen receptor alpha gene polymorphisms with bone mineral density in postmenopausal Indian women.

Bone mineral density (BMD) is the major determinant of osteoporotic fracture risk with a particular genetic background. However, consensus on the association of BMD with specific gene locus has not been reached. In the present study, we investigated the potential association of estrogen receptor alpha (ER alpha) gene intron I polymorphisms with BMD in 246 postmenopausal Indian women (average age 54.2+/-3.4 years). All the subjects were genotyped for XbaI and PvuII polymorphisms and underwent BMD measurements at spine and hip by dual energy X-ray absorptiometery. The average BMD of subjects with the genotypes XX and PP (absence of restriction sites for XbaI and PvuII, respectively) was 12.7 and 5.4% higher at the spine and 13.1 and 4.6% higher at the hip, respectively, than those with genotypes xx and pp. In age vs. BMD scatterplot, the intercept and slope of regression lines for genotypes xx and pp at spine and hip demonstrated comparatively rapid decrease in BMD across the age. The genotype XX was significantly prevalent (p<0.001) in women with normal bone mass (32%) and genotype xx in women with osteoporotic bone mass (35.3%), within the group. A significantly higher relative risk was associated with xx genotype. The study concludes that genetic variations at ER alpha gene locus, perhaps, are associated with BMD in Indian women and may influence some determinant of bone metabolism resulting in accelerated bone loss with age.

Development and validation of an ELISA for hemoglobin-A2: a novel method for beta-thalassemia screening in developing countries.

Hemoglobin-A2 (HbA2) measurement in human hemolysates has great significance, since its level can indicate beta-thalassemia carrier status in otherwise healthy individuals. An ELISA for HbA2 using antiserum monospecific to the delta chain of HbA2 and affinity purified antirabbit gamma globulins (ARGG) conjugated to horseradish peroxidase (HRP) have been developed. The monospecific antiserum used does not cross react with other hemoglobins. Hemolysates from volunteers are used for measurement of HbA2. In a limited trial for beta-thalassemia carrier screening (n = 350), the results obtained with the developed ELISA are comparable with those obtained with a micro-column chromatography method (r > or = 0.89). The developed ELISA is simple, accurate, precise, inexpensive, and several samples can be processed simultaneously with ease, making this system a suitable candidate for transforming into a user friendly kit.