Hormone-stimulated modulation of endocytic trafficking in osteoclasts

Copyright @ 2012 Stenbeck, Lawrence and Albert. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. This article has been made available through the Brunel Open Access Publishing Fund.A precise control of vesicular trafficking is crucial not only for osteoclastic bone resorption, but also for the crosstalk between osteoclasts and osteoblasts, which regulates bone homeostasis. In addition to the release of growth factors and modulators, such as glutamate, flux through the intracellular trafficking routes could also provide the osteoclast with a monitoring function of its resorption activity. To establish the signaling pathways regulating trafficking events in resorbing osteoclasts, we used the bone conserving hormone calcitonin, which has the unique property of inducing osteoclast quiescence. Calcitonin acts through the calcitonin receptor and activates multiple signaling pathways. By monitoring trafficking of a fluorescent low molecular weight probe in mature, bone resorbing osteoclasts we show for the first time that calcitonin blocks endocytosis from the ruffled border by phospholipase C (PLC) activation. Furthermore, we identify a requirement for polyunsaturated fatty acids in endocytic trafficking in osteoclasts. Inhibition of PLC prior to calcitonin treatment restores endocytosis to 75% of untreated rates. This effect is independent of protein kinase C activation and can be mimicked by an increase in intracellular calcium. We thus define an essential role for intracellular calcium levels in the maintenance of endocytosis in osteoclasts.Arthritis Research UK Grant (18197)

Insights into Activation Mechanisms of Store-Operated TRPC1 Channels in Vascular Smooth Muscle.

In vascular smooth muscle cells (VMSCs), the stimulation of store-operated channels (SOCs) mediate Ca2+ influx pathways which regulate important cellular functions including contraction, proliferation, migration, and growth that are associated with the development of vascular diseases. It is therefore important that we understand the biophysical, molecular composition, activation pathways, and physiological significance of SOCs in VSMCs as these maybe future therapeutic targets for conditions such as hypertension and atherosclerosis. Archetypal SOCs called calcium release-activated channels (CRACs) are composed of Orai1 proteins and are stimulated by the endo/sarcoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) following store depletion. In contrast, this review focuses on proposals that canonical transient receptor potential (TRPC) channels composed of a heteromeric TRPC1/C5 molecular template, with TRPC1 conferring activation by store depletion, mediate SOCs in native contractile VSMCs. In particular, it summarizes our recent findings which describe a novel activation pathway of these TRPC1-based SOCs, in which protein kinase C (PKC)-dependent TRPC1 phosphorylation and phosphatidylinositol 4,5-bisphosphate (PIP2) are obligatory for channel opening. This PKC- and PIP2-mediated gating mechanism is regulated by the PIP2-binding protein myristoylated alanine-rich C kinase (MARCKS) and is coupled to store depletion by TRPC1-STIM1 interactions which induce Gq/PLCβ1 activity. Interestingly, the biophysical properties and activation mechanisms of TRPC1-based SOCs in native contractile VSMCs are unlikely to involve Orai1

Store-operated interactions between plasmalemmal STIM1 and TRPC1 proteins stimulate PLCβ1 to induce TRPC1 channel activation in vascular smooth muscle cells.

KEY POINTS: Depletion of Ca(2+) stores activates store-operated channels (SOCs), which mediate Ca(2+) entry pathways that regulate cellular processes such as contraction, proliferation and gene expression. In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques. Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1(-/-) cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline. These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating. ABSTRACT: In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca(2+) entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca(2+) sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1(-/-) VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1(-/-) cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating

Factors Associated with Unmet Need for Modern Contraception in Post-Conflict Liberia

We examined the association between intimate partner violence and  unmet need for modern contraception in post-conflict Liberia. This is a secondary analysis of data collected using the Priorities for Local AIDS Control Efforts (PLACE) method. Data from 499 sexually experienced young women (aged 14-25) in Montserrado County, Liberia were examined.  Intimate partner violence (55.7%), unintended pregnancy (83.2%), and abortion (45.3%) were pervasive in the study population. An estimated 35.9% of respondents had an unmet need for modern contraception.  However, multivariate logistic regression results did not reveal an  association between intimate partner violence and unmet need (OR 1.11; 95% CI 0.70-1.75). Among covariates examined, only contraceptive use at sexual debut (26.1%) was significantly associated with unmet need (OR 0.27; 95% CI 0.14-0.52). Liberian youth need information about and access to modern contraceptive methods besides condoms. Interventions to identify and treat victims of violence are also needed. Afr J Reprod Health 2014; 18[2]: 58-67).Key words: Intimate partner violence, unintended pregnancy, abortion, unmet need for contraception, adolescents, Liberi

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells.

Ca(2+)-permeable store-operated channels (SOCs) mediate Ca(2+) entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1(-/-) mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1(-/-) VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1(-/-) VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1(-/-) VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype

Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced nitric oxide production and vasorelaxation in rabbit mesenteric arteries.

Stimulation of calcium-sensing receptors (CaSR) by increasing the external calcium concentration (Ca(2+)]o) induces endothelium-dependent vasorelaxation through nitric oxide (NO) production and activation of intermediate Ca(2+)-activated K(+) currents (IKCa) channels in rabbit mesenteric arteries. The present study investigates the potential role of heteromeric TRPV4-TRPC1 channels in mediating these CaSR-induced vascular responses. Immunocytochemical and proximity ligation assays showed that TRPV4 and TRPC1 proteins were expressed and co-localised at the plasma membrane of freshly isolated endothelial cells (ECs). In wire myography studies, increasing [Ca(2+)]o between 1 and 6mM induced concentration-dependent relaxations of methoxamine (MO)-induced pre-contracted tone, which were inhibited by the TRPV4 antagonists RN1734 and HC067047, and the externally-acting TRPC1 blocking antibody T1E3. In addition, CaSR-evoked NO production in ECs measured using the fluorescent NO indicator DAF-FM was reduced by RN1734 and T1E3. In contrast, [Ca(2+)]o-evoked perforated-patch IKCa currents in ECs were unaffected by RN1734 and T1E3. The TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent relaxation of MO-evoked pre-contracted tone and increased NO production, which were inhibited by the NO synthase inhibitor L-NAME, RN1734 and T1E3. GSK activated 6pS cation channel activity in cell-attached patches from ECs which was blocked by RN1734 and T1E3. These findings indicate that heteromeric TRPV4-TRPC1 channels mediate CaSR-induced vasorelaxation through NO production but not IKCa channel activation in rabbit mesenteric arteries. This further implicates CaSR-induced pathways and heteromeric TRPV4-TRPC1 channels in regulating vascular tone

MARCKS mediates vascular contractility through regulating interactions between voltage-gated Ca2+ channels and PIP2.

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as substrate and unmodified ligand for Gq-protein-coupled receptor signalling in vascular smooth muscle cells (VSMCs) that is central for initiating contractility. The present work investigated how PIP2 might perform these two potentially conflicting roles by studying the effect of myristoylated alanine-rich C kinase substrate (MARCKS), a PIP2-binding protein, on vascular contractility in rat and mouse mesenteric arteries. Using wire myography, MANS peptide (MANS), a MARCKS inhibitor, produced robust contractions with a pharmacological profile suggesting a predominantly role for L-type (CaV1.2) voltage-gated Ca2+ channels (VGCC). Knockdown of MARCKS using morpholino oligonucleotides reduced contractions induced by MANS and stimulation of α1-adrenoceptors and thromboxane receptors with methoxamine (MO) and U46619 respectively. Immunocytochemistry and proximity ligation assays demonstrated that MARCKS and CaV1.2 proteins co-localise at the plasma membrane in unstimulated tissue, and that MANS and MO reduced these interactions and induced translocation of MARCKS from the plasma membrane to the cytosol. Dot-blots revealed greater PIP2 binding to MARCKS than CaV1.2 in unstimulated tissue, with this binding profile reversed following stimulation by MANS and MO. MANS evoked an increase in peak amplitude and shifted the activation curve to more negative membrane potentials of whole-cell voltage-gated Ca2+ currents, which were prevented by depleting PIP2 levels with wortmannin. This present study indicates for the first time that MARCKS is important regulating vascular contractility and suggests that disinhibition of MARCKS by MANS or vasoconstrictors may induce contraction through releasing PIP2 into the local environment where it increases voltage-gated Ca2+ channel activity

Fetal and infant growth and the risk of obesity during early childhood: the Generation R Study.

OBJECTIVE: To examine whether infant growth rates are influenced by fetal growth characteristics and are associated with the risks of overweight and obesity in early childhood. DESIGN: This study was embedded in the Generation R Study, a population-based prospective cohort study from fetal life onward. METHODS: Fetal growth characteristics (femur length (FL) and estimated fetal weight (EFW)) were assessed in the second and third trimesters and at birth (length and weight). Infant peak weight velocity (PWV), peak height velocity (PHV), and body mass index at adiposity peak (BMIAP) were derived for 6267 infants with multiple height and weight measurements. RESULTS: EFW measured during the second trimester was positively associated with PWV and BMIAP during infancy. Subjects with a smaller weight gain between the third trimester and birth had a higher PWV. FL measured during the second trimester was positively associated with PHV. Gradual length gain between the second and third trimesters and between the third trimester and birth were associated with higher PHV. Compared with infants in the lowest quintile, the infants in the highest quintile of PWV had strongly increased risks of overweight/obesity at the age of 4 years (odds ratio (95% confidence interval): 15.01 (9.63, 23.38)). CONCLUSION: Fetal growth characteristics strongly influence infant growth rates. A higher PWV, which generally occurs in the first month after birth, was associated with an increased risk of overweight and obesity at 4 years of age. Longer follow-up studies are necessary to determine how fetal and infant growth patterns affect the risk of disease in later life

Obligatory role for PKCδ in PIP2 -mediated activation of store-operated TRPC1 channels in vascular smooth muscle cells.

KEY POINTS: In vascular smooth muscle cells (VSMCs), activation of Ca2+ -permeable store-operated channels (SOCs) composed of canonical transient receptor potential channel 1 (TRPC1) subunits mediate Ca2+ entry pathways which regulate contraction, proliferation and migration that are processes associated with vascular disease. Activation of TRPC1-based SOCs requires protein kinase C (PKC) activity, which is proposed to phosphorylate TRPC1 proteins to promote channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). We investigated the identity of the PKC isoform involved in activating TRPC1-based SOCs in rat mesenteric artery VSMCs. TRPC1-based SOCs were reduced by PKCδ inhibitors and knockdown of PKCδ expression. Store depletion induced interactions between TRPC1 and PKCδ and PKCδ-dependent phosphorylation of TRPC1. Furthermore, generation of store-operated interactions between PIP2 and TRPC1 and activation of TRPC1-based SOCs by PIP2 required PKCδ. These findings reveal that PKCδ activity has an obligatory role in activating TRPC1-based SOCs, through regulating PIP2 -mediated channel opening. ABSTRACT: In vascular smooth muscle cells (VMSCs), stimulation of Ca2+ -permeable canonical transient receptor potential channel 1 (TRPC1)-based store-operated channels (SOCs) mediate Ca2+ entry pathways which regulate cell contraction, proliferation and migration that are processes associated with vascular disease. It is therefore important to understand how TRPC1-based SOCs are activated. Stimulation of TRPC1-based SOCs requires protein kinase C (PKC) activity, with store-operated PKC-dependent phosphorylation of TRPC1 essential for channel opening by phosphatidylinositol 4,5-bisphosphate (PIP2 ). Experimental protocols used to activate TRPC1-based SOCs suggest that the PKC isoform involved requires diacylglycerol (DAG) but is Ca2+ -insensitive, which are characteristics of the novel group of PKC isoforms (δ, ε, η, θ). Hence the present study examines if a novel PKC isoform(s) is involved in activating TRPC1-based SOCs in contractile rat mesenteric artery VSMCs. Store-operated whole-cell cation currents were blocked by Pico145, a highly selective and potent TRPC1/4/5 channel blocker and T1E3, a TRPC1 blocking antibody. PKCδ was expressed in VSMCs, and selective PKCδ inhibitory peptides and knockdown of PKCδ expression with morpholinos oligomers inhibited TRPC1-based SOCs. TRPC1 and PKCδ interactions and phosphorylation of TRPC1 induced by store depletion were both reduced by pharmacological inhibition and PKCδ knockdown. In addition, store-operated PIP2 and TRPC1 interactions were blocked by PKCδ inhibition, and PKCδ was required for PIP2 -mediated activation of TRPC1 currents. These results identify involvement of PKCδ in stimulation of TRPC1-based SOCs and highlights that store-operated PKCδ activity is obligatory for channel opening by PIP2 , the likely activating ligand. This article is protected by copyright. All rights reserved

Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling.

Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes