Membrane Caveolae Research Articles

Sheath Cell Invasion and Trans-differentiation Repair Mechanical Damage Caused by Loss of Caveolae in the Zebrafish Notochord

The notochord, a conserved axial structure required for embryonic axis elongation and spine development, consists of giant vacuolated cells surrounded by an epithelial sheath [1-3]. During morphogenesis, vacuolated cells maintain their structural integrity despite being under constant mechanical stress [4]. We hypothesized that the high density of caveolae present in vacuolated cells [5, 6] could buffer mechanical tension. Caveolae are 50- to 80-nm membrane invaginations lined by cage-like polygonal structures [7, 8] formed by caveolin 1 (Cav1) or Cav3 and one of the cavin proteins [6, 9-11]. Recent invitro work has shown that plasma membrane caveolae constitute a membrane reservoir that can buffer mechanical stresses such as stretching or osmotic swelling [12]. Moreover, mechanical integrity of vascular and muscle cells is partly dependent on caveolae [13-15]. However, the invivo mechano-protective roles of caveolae have only begun to be explored. Using zebrafish mutants for cav1, cav3, and cavin1b, we show that caveolae are essential for notochord integrity. Upon loss of caveola function, vacuolated cells collapse at discrete positions under the mechanical strain of locomotion. Then, sheath cells invade the inner notochord and differentiate into vacuolated cells, thereby restoring notochord function and allowing normal spine development. Our data further indicate that nucleotides released by dying vacuolated cells promote sheath cell vacuolization and trans-differentiation. This work reveals a novel structural role for caveolae in vertebrates and provides unique insights into the mechanisms that safeguard notochord and spine development.

The expression of the rare caveolin-3 variant T78M alters cardiac ion channels function and membrane excitability.

AimsCaveolinopathies are a family of genetic disorders arising from alterations of the caveolin-3 (cav-3) gene. The T78M cav-3 variant has been associated with both skeletal and cardiac muscle pathologies but its functional contribution, especially to cardiac diseases, is still controversial. Here, we evaluated the effect of the T78M cav-3 variant on cardiac ion channel function and membrane excitability.Methods and resultsWe transfected either the wild type (WT) or T78M cav-3 in caveolin-1 knock-out mouse embryonic fibroblasts and found by immunofluorescence and electron microscopy that both are expressed at the plasma membrane and form caveolae. Two ion channels known to interact and co-immunoprecipitate with the cav-3, hKv1.5 and hHCN4, interact also with T78M cav-3 and reside in lipid rafts. Electrophysiological analysis showed that the T78M cav-3 causes hKv1.5 channels to activate and inactivate at more hyperpolarized potentials and the hHCN4 channels to activate at more depolarized potentials, in a dominant way. In spontaneously beating neonatal cardiomyocytes, the expression of the T78M cav-3 significantly increased action potential peak-to-peak variability without altering neither the mean rate nor the maximum diastolic potential. We also found that in a small cohort of patients with supraventricular arrhythmias, the T78M cav-3 variant is more frequent than in the general population. Finally, in silico analysis of both sinoatrial and atrial cell models confirmed that the T78M-dependent changes are compatible with a pro-arrhythmic effect.ConclusionThis study demonstrates that the T78M cav-3 induces complex modifications in ion channel function that ultimately alter membrane excitability. The presence of the T78M cav-3 can thus generate a susceptible substrate that, in concert with other structural alterations and/or genetic mutations, may become arrhythmogenic.

TIR/BB-loop mimetic AS-1 attenuates cardiac ischemia/reperfusion injury via a caveolae and caveolin-3-dependent mechanism

AS-1, the TIR/BB loop mimetic, plays a protective role in cardiac ischemia/reperfusion (I/R) but the molecular mechanism remains unclear. The muscle specific caveolin3 (Cav-3) and the caveolae have been found to be critical for cardioprotection. This study aimed to evaluate our hypothesis that caveolae and Cav-3 are essential for AS-1-induced cardioprotection against myocardial I/R injury. To address these issues, we analyzed the involvement of Cav-3 in AS-1 mediated cardioprotection both in vivo and in vitro. We demonstrate that AS-1 administration significantly decreased infarct size, improved cardiac function after myocardial I/R and modulated membrane caveolae and Cav-3 expression in the myocardium. For in vitro studies, AS-1 treatment prevented Cav-3 re-distribution induced by H/R injury. In contrast, disruption of caveolae by MCD treatment or Cav-3 knockdown abolished the protection against H/R-induced myocytes injury by AS-1. Our findings reveal that AS-1 attenuates myocardial I/R injury through caveolae and Cav-3 dependent mechanism.

Follicular Stimulating Hormone Accelerates Atherogenesis by Increasing Endothelial VCAM-1 Expression

Rationale: Postmenopausal atherosclerosis (AS) has for decades been attributed to estrogen deficiency. Although the follicular stimulating hormone (FSH) levels rise sharply in parallel, the direct effect of FSH on AS has never been investigated. In this study, we explored the possible role of FSH in the development of AS.Methods: This was a prospective cohort study of 48 healthy premenopausal and 15 postmenopausal women. ApoE knockout mice were used as atherosclerosis model and human umbilical vascular endothelial cells (HUVECs) were cultured as cell model. Serum hormones and vascular cell adhesion molecule-1 (VCAM-1) levels were measured. Real-time PCR, histology for atherosclerotic lesions, immunofluorescence, luciferase assay, transfection experiments, flow chamber adhesion assay and western blot were performed.Results: In ApoE knockout mice, administration of FSH increased the atherosclerotic lesions and serum VCAM-1 concentration. Importantly, in blood samples of postmenopausal women, we detected significantly higher levels of FSH and VCAM-1 compared with those from premenopausal women, and there was a positive correlation between these two molecules. In cultured HUVECs, FSH receptor (FSHR) mRNA and protein expression were detected and FSH enhanced VCAM-1 expression. This effect was mediated by the activation of nuclear factor κB (NF-κB), which was sequentially enhanced by the activation of PI3K/Akt/mTOR cascade. FSH first enhanced GαS activity resulting in elevated cAMP level and PKA activity, which relayed the signals from FSHR to the PI3K/Akt/mTOR cascade. Furthermore, FSHR was detected in endothelial caveolae fraction and interacted with caveolin-1 and GαS. The disruption of caveolae or the silencing of caveolin-1 blocked FSH effects on signaling activation and VCAM-1 expression, suggesting the existence of a functional signaling module in membrane caveolae. Finally, FSH increased human monocyte adhesion to HUVECs which was reversed by the VCAM-1 neutralizing antibody.Conclusion: FSHR was located in the membrane caveolae of HUVECs and FSH promoted VCAM-1 expression via FSHR/GαS /cAMP/PKA and PI3K/Akt/mTOR/NF-κB pathway. This may contribute to the deleterious role of FSH in the development of AS in postmenopausal women.

Caveolins and caveolae in ocular physiology and pathophysiology.

Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function.

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.

Attenuated rapid onset vasodilation with greater force production in skeletal muscle of caveolin-2-/- mice.

Caveolin-2 (Cav2) is a major protein component of caveolae in membranes of vascular smooth muscle and endothelium, yet its absence alters the ultrastructure of skeletal muscle fibers. To gain insight into Cav2 function in skeletal muscle, we tested the hypothesis that genetic deletion of Cav2 would alter microvascular reactivity and depress contractile function of skeletal muscle in vivo. In the left gluteus maximus muscle (GM) of anesthetized Cav2(-/-) and wild-type (WT) male mice (age, 6 mo), microvascular responses to physiological agonists and to GM contractions were studied at 34°C. For feed arteries (FA), first- (1A), second- (2A) and third-order (3A) arterioles, respective mean diameters at rest (45, 35, 25, 12 μm) and during maximal dilation (65, 55, 45, 30 μm) were similar between groups. Cumulative dilations to ACh (10(-9) to 10(-5) M) and constrictions to norepinephrine (10(-9) to 10(-5) M) were also similar between groups, as were steady-state dilations during rhythmic twitch contractions (2 and 4 Hz; 30 s). For single tetanic contractions (100 Hz; 100, 250, and 500 ms), rapid onset vasodilation (ROV) increased with contraction duration throughout networks in GM of both groups but was reduced by nearly half in Cav2(-/-) mice compared with WT mice (P < 0.05). Nevertheless, maximal force during tetanic contraction was ∼40% greater in GM of Cav2(-/-) vs. WT mice (152 ± 14 vs. 110 ± 3 mN per square millimeter, respectively; P < 0.05). Thus, while structural and functional properties of resistance networks are well maintained in the GM of Cav2(-/-) mice, diminished ROV with greater force production reveals novel physiological roles for Cav2 in skeletal muscle.

Increase in caveolae and caveolin-1 expression modulates agonist-induced contraction and store- and receptor-operated Ca2 + entry in pulmonary arteries of pulmonary hypertensive rats

Caveolin-1 (Cav-1) is a major component protein associated with caveolae in the plasma membrane and has been identified as a regulator of store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ entry (ROCE). However, the contributions of caveolae/Cav-1 of pulmonary arterial smooth muscle cells (PASMCs) to the altered Ca2+ signaling pathways in pulmonary arteries (PAs) during pulmonary hypertension (PH) have not been fully characterized. The present study quantified caveolae number and Cav-1 expression, and determined the effects of caveolae disruption on ET-1, cyclopiazonic acid (CPA) and 1-Oleoyl-2-acetyl-glycerol (OAG)-induced contraction in PAs and Ca2+ influx in PASMCs of chronic hypoxia (CH)- and monocrotaline (MCT)-induced PH rats. We found that the number of caveolae, and the Cav-1 mRNA and protein levels were increased significantly in PASMCs in both PH models. Disruption of caveolae by cholesterol depletion with methyl-β-cyclodextrin (MβCD) significantly inhibited the contractile response to ET-1, CPA and OAG in PAs of control rats. ET-1, SOCE and ROCE-mediated contractile responses were enhanced, and their susceptibility to MβCD suppression was potentiated in the two PH models. MβCD-induced inhibition was reversed by cholesterol repletion. Introduction of Cav-1 scaffolding domain peptide to mimic Cav-1 upregulation caused significant increase in CPA- and OAG-induced Ca2+ entry in PASMCs of control, CH and MCT-treated groups. Our results suggest that the increase in caveolae and Cav-1 expression in PH contributes to the enhanced agonist-induced contraction of PA via modulation of SOCE and ROCE; and targeting caveolae/Cav-1 in PASMCs may provide a novel therapeutic strategy for the treatment of PH.

Activation of TRPV2 and BKCa channels by the LL-37 enantiomers stimulates calcium entry and migration of cancer cells.

Expression of the antimicrobial peptide hCAP18/LL-37 is associated to malignancy in various cancer forms, stimulating cell migration and metastasis. We report that LL-37 induces migration of three cancer cell lines by activating the TRPV2 calcium-permeable channel and recruiting it to pseudopodia through activation of the PI3K/AKT pathway. Ca2+ entry through TRPV2 cooperated with a K+ efflux through the BKCa channel. In a panel of human breast tumors, the expression of TRPV2 and LL-37 was found to be positively correlated. The D-enantiomer of LL-37 showed identical effects as the L-peptide, suggesting that no binding to a specific receptor was involved. LL-37 attached to caveolae and pseudopodia membranes and decreased membrane fluidity, suggesting that a modification of the physical properties of the lipid membrane bilayer was the underlying mechanism of its effects.

Modulation of 17β‐Estradiol Signaling on Cellular Proliferation by Caveolin‐2

The sex hormone 17β-estradiol (E2) exerts pleiotropic effects by binding to the ligand-activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects (i.e., estrogen responsive element [ERE]-based gene transcription) and non-transcriptional membrane-initiated effects (i.e., the activation of extra-nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω-shaped membrane sub-domains that are composed of scaffold proteins named caveolins (i.e., caveolin-1, caveolin-2, and caveolin-3). Although caveolin-3 is exclusively expressed in muscles, caveolin-1 and caveolin-2 are co-expressed in all human tissues. From a functional point of view, caveolin-2 can operate both dependently on and independently of caveolin-1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin-1 and ERα has been reported in the control of E2-induced physiological effects, the role of caveolin-2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA-mediated caveolin-2 depletion in breast ductal carcinoma cells (MCF-7) reduces E2-induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα-mediated extra-nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin-1 depletion on ERα signaling in MCF-7 cells is shown to be similar to that elicited by siRNA-mediated caveolin-2 depletion. Altogether, these data demonstrate that caveolin-2 expression is necessary for the control of E2-dependent cellular proliferation.

Ionizing Radiation Induces Phosphorylation of Caveolin-1 on Tyr14

Caveolin-1 (CAV1) is a 22 kDa membrane protein associated with caveolae in the cell membrane. It is involved in endocytosis, modulation of signal transduction, and has been reported to mediate radioprotection. Various stimuli such as oxidative stress, insulin, or osmotic shock are known to induce phosphorylation of caveolin-1 on Tyr14. This event is an important prerequisite for CAV1 function in the formation of caveolae and the interaction with multiple signaling molecules. The aim of the present study was to test if ionizing radiation (IR) induces phosphorylation of CAV1 on Tyr14 similar to oxidative stress, and to test the role of stress signaling kinases p38, Src family kinases (SFK), and c-Jun N-terminal kinase (JNK). The human lymphoblastoid cell line TK6, which does not express endogenous CAV1, was transduced with two different lentiviral vectors, containing either the cDNA of wtCAV1 or the cDNA of CAV1Y14F (substitution of Tyr by Phe on position 14) connected by the IRES element of EMCV with the cDNA of EGFP. Oxidative stress in TK6-CAV1 and TK6-CAV1Y14F was induced by treatment with 1 mM - 50 mM H2O2 for 20 min at 37 °C; cell irradiation was performed using 6 MV X-ray (6.7 Gy/min, Elekta Synergy). Cell lysates were prepared at different time points (5 min to 8 h) after IR. Specific kinase inhibitors were used to explore the functional relevance of kinase activation for CAV1 phosphorylation after H2O2 or IR. Phosphorylation was detected by Western blotting using phospho-specific antibodies, quantified and normalized to the unphosphorylated form. Mean values and SEM of at least three independent experiments were calculated. Phosphorylation of CAV1, p38, SFK and JNK in TK6-CAV1 was markedly increased by H2O2 concentrations in the range of 5 mM – 20 mM. Activation of p38, SFK and JNK was similar in TK6-CAV1Y14F and thus independent of Tyr14 phosphorylation of CAV1. Radiation doses of 2 Gy and 4 Gy clearly increased phosphorylation of CAV1, p38, SFK and JNK with different kinetics. While maximum CAV1 and SFK phosphorylation occurred early after irradiation (15 min – 1 h), activation of p38 and JNK peaked at the later time points (1 h – 3 h). Inhibition of p38, SFK and JNK revealed SFK as the major kinases for CAV1 phosphorylation on Tyr14 after induction of oxidative stress. Induction of CAV1 phosphorylation on Tyr14 and activation of stress-associated signaling molecules indicated the activation of similar signaling proteins in response to H2O2 treatment and IR with H2O2 -induced phosphorylation being more potent. Activation of SFK was an important factor for the phosphorylation of CAV1 after treatment with H2O2 in the TK6 cells used here. Studies on the role of the different kinases and phosphorylation of CAV1 regarding proliferation and apoptosis after irradiation are in progress.

Caveolin-1 in Cardiovascular Disease: A Double-Edged Sword.

Endothelial dysfunction, as manifested by an attenuation of nitric oxide (NO)–mediated vasodilation, is recognized to be a fundamental abnormality in the genesis of hypertension, atherosclerosis, and coronary artery disease (1). Metabolic risk factors, such as obesity, insulin resistance, and type 2 diabetes (T2D), can initiate and accelerate endothelial dysfunction leading to cardiovascular disease (CVD) (1). Endothelial dysfunction in association with metabolic abnormalities is typically caused by a combination of reduced production and increased destruction of NO leading to a decrease in NO bioavailability (1,2). NO production in response to various factors, such as increased shear stress, is mediated by endothelial nitric oxide synthase (eNOS), which is constitutively expressed in endothelial cells (ECs) and is tightly controlled by various membrane-bound receptors and regulatory proteins under physiological conditions (3). Caveolin-1 (Cav-1), an anchoring protein in the plasma membrane caveolae in ECs and vascular smooth muscle cells (VSMCs), attenuates endothelial NO production by occupying the calcium/calmodulin (Ca2+/CaM) binding site of eNOS (4) (Fig. 1). Increases in caveolin and eNOS interaction, as may occur with hyperlipidemia, reduce NO production and promote endothelial dysfunction and atherosclerotic lesion formation. This process is mediated by increased lipoprotein trafficking across the vascular endothelium (5,6). Therefore, treating hyperlipidemia as an early intervention to help …

Tanshinone II A, a multiple target neuroprotectant, promotes caveolae-dependent neuronal differentiation

Neuron loss is one fundamental features of neurodegenerative diseases. Stimulating endogenous neurogenesis, especially neuronal differentiation, might potentially provide therapeutic effects to these diseases. In this study, tanshinone II A (TIIA), a multiple target neuroprotectant, was demonstrated to promote dose-dependent neuronal differentiation in three cell models of immortalized C17.2 neuronal stem cells, rat embryonic cortical neural stem cells (NSCs) and rat PC12 pheochromocytoma cells. In particular, TIIA exerted promising effects on NSCs even at the dose of 3nM. In PC12 cells, TIIA activated mitogen-activated protein kinase 42/44 (MAPK42/44) and its downstream transcription factor, cAMP response element-binding protein (CREB). In addition, TIIA up-regulated the expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The MEK inhibitor and the antagonist to the receptors of NGF and BDNF could partially attenuate the differentiation effects, indicating that MAPK42/44 mediated BDNF and NGF signals were involved in TIIA's differentiation effects. Caveolin-1 (CAV-1), the major functional protein of membrane caveolae, plays critical roles in the endocytosis of exogenous materials. CAV1, which was activated by TIIA, might help TIIA transport across cell membrane to initiate its differentiation effects. It was proven by the evidences that suppressing the function of caveolin inhibited the differentiation effects of TIIA. Therefore, we concluded that TIIA promoted neuronal differentiation partially through MAPK42/44 mediated BDNF and NGF signals in a caveolae-dependent manner.

Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes

Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSμ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095–1289 and Santa Cruz Biotechnology aa 2–300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was ∼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSβ. RT-PCR results confirmed the expressions of both nNOSα and nNOSβ mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. ∼140 kDa), confirming nNOSβ. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS−/− mice (which lacks nNOSα/μ). Furthermore, nNOSβ expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of ∼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.

Abstract 722: Caveolin-1-dependent endocytosis enhanced chemosensitivity of TDM-1 in HER-2-positive breast cancer cells

Abstract Trastuzumab emtansine (TDM-1, kadcyla) is the antibody drug conjugate (ADC) was approved to treat HER-2 positive metastatic breast cancer. Although its effectiveness, however, the biomarker of drug resistance of TDM-1 has not been identified. The efficacy of ADC is restricted to the antibody internalization in the cell; therefore, the drug sensitivity may be associated with cellular vesicle trafficking system. Caveolin-1, which is the 21 KD membrane protein that is required for caveolae structure formation and membrane endocytic transport. In this study, the relationship of caveolin-1 expression and the chemosensitivity of TDM-1 in HER-2 positive breast cancer cells was investigated. Immunohistochemical staining of 32 specimens of breast cancer patients were evaluated for caveolin-1 expression. For in vitro study, BT-474 and SKBR-3 breast cancer cells which represent different caveolin-1 expression were treated with trastuzumab or T-DM1. Cell viability and molecular localization were examined. Cellular caveolin-1 expression was also manipulated by transfection of GFP tagged caveolin-1 or caveolin-1 shRNA plasmid, cell viability was then determined after drug treatment. In the results, Immunohistochemical study showed 68% (22/32) overexpression of Caveolin-1 in breast cancerous specimens. And eight cases with high expression of Caveolin-1 were found in HER-2 positive breast cancerous patients (72.1%, 8/11). In cell biology study, T-DM1 was found colocalized with caveolin-1 by confocal microscopy. SKBR-3 which represents higher caveolin-1 expression than BT-474 was more sensitive to T-DM1 treatment. After molecular transfection, BT-474 cells with overexpression of caveolin-1 increased chemosensitivity in the treatment of TDM-1, instead, SKBR-3 cells transfected with caveolin-1 shRNA decreased T-DM1 sensitivity. Expression of caveolin-1 could mediate endocytosis and promoting the internalization of TDM-1 into HER-2 positive cancer cells. Thereafter, caveolin-1 may be an effective predictor for TDM-1 treatment in the future. Citation Format: Yuan-Chiang Chung, Wan-Chen Wei, King-Jen Chang, Wei-Ting Chao. Caveolin-1-dependent endocytosis enhanced chemosensitivity of TDM-1 in HER-2-positive breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 722. doi:10.1158/1538-7445.AM2015-722

Pathogenic mechanisms of lung adenocarcinoma in smokers and non-smokers determined by gene expression interrogation

Cigarette smoking is the leading risk factor for lung cancer, which accounts for the highest number of cancer-related mortalities worldwide in men and women. Individuals with a history of smoking are 15-30 times more likely to develop lung cancer compared with those who do not smoke. However, our understanding of the underlying molecular mechanisms that contribute to lung tumorigenesis in smokers versus non-smokers remains incomplete. In order to investigate such mechanisms, the present study aimed to systemically interrogate microarray datasets from tumor biopsies and matching normal tissues from stage I and II lung adenocarcinoma patients who had never smoked or were current smokers. The gene expression analysis identified 422 (99 upregulated and 323 downregulated) and 534 (174 upregulated and 360 downregulated) differentially-expressed genes from the never-smokers and current smokers, respectively, and the two groups shared 277 genes that exhibited similar trends of alteration. These genes encode regulators that are involved in a variety of cellular functions, including collagen metabolism and homeostasis of caveolae plasma membranes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes characterization indicated that biological pathways, including extracellular matrix-receptor interaction and cell migration and proliferation, were all affected in the lung cancer patients regardless of the smoking status. However, smoking induced a unique gene expression pattern characterized by upregulation of cell cycle regulators (CDK1, CCNB1 and CDC20), as well as significantly affected biological networks, including p53 signaling pathways. Taken together, these findings suggest novel mechanistic insights, and provide an improved understanding of the smoking-induced molecular alterations that contribute to the pathogenesis of lung adenocarcinoma.

PTRF is associated with caveolin 1 at the time of receptivity: but SDPR is absent at the same time.

The plasma membrane of uterine epithelial cells undergoes a number of changes during early pregnancy. The changes in the basolateral membrane at the time of implantation in particular change from being smooth to highly tortuous in morphology, along with a dramatic increase in the number of morphological caveolae at this time. The major protein of caveolar membranes is caveolin, and previous studies have shown that RNA pol I transcription factor (PTRF) and serum deprivation protein response (SDPR) are the two members of the cavin protein family. These proteins are known to be involved in caveolae biogenesis, where they directly bind to cholesterol and lipids and have been reported to promote membrane curvature. As there is an increase in membrane tortuosity and caveolae at the time of implantation, this study investigated PTRF and SDPR to explore the possible roles that they play in the morphology of the uterine epithelium during early pregnancy. PTRF protein abundance did not change in uterine epithelial cells during early pregnancy or in response to ovarian hormones. At the time of implantation in uterine epithelial cells, PTRF co-immunoprecipitated with caveolin 1, thereby demonstrating an association with caveolin-1 at the basal plasma membrane in caveolae. SDPR protein was observed to be present only at the time of fertilisation, and also under the influence of oestrogen alone, where a cytoplasmic localisation in uterine epithelial cells was observed. The localisation and expression PTRF and SDPR in uterine epithelial cells during early pregnancy suggest that they have roles in the maintenance of lipids and cholesterol in the plasma membrane. PTRF and lack of SDPR may contribute not only to the morphology of the basal plasma membrane as observed at the time of implantation, but also to the maintenance of epithelial polarity during early pregnancy.

Characterization of the dystrophin-glycoprotein complex in airway smooth muscle: role of δ-sarcoglycan in airway responsiveness.

The dystrophin-glycoprotein complex (DGC) is an integral part of caveolae microdomains, and its interaction with caveolin-1 is essential for the phenotype and functional properties of airway smooth muscle (ASM). The sarcoglycan complex provides stability to the dystroglycan complex, but its role in ASM contraction and lung physiology in not understood. We tested whether δ-sarcoglycan (δ-SG), through its interaction with the DGC, is a determinant of ASM contraction ex vivo and airway mechanics in vivo. We measured methacholine (MCh)-induced isometric contraction and airway mechanics in δ-SG KO and wild-type mice. Last, we performed immunoblotting and transmission electron microscopy to assess DGC protein expression and the ultrastructural features of tracheal smooth muscle. Our results reveal an age-dependent reduction in the MCh-induced tracheal isometric force and significant reduction in airway resistance at high concentrations of MCh (50.0 mg/mL) in δ-SG KO mice. The changes in contraction and lung function correlated with decreased caveolin-1 and β-dystroglycan abundance, as well as an age-dependent loss of caveolae in the cell membrane of tracheal smooth muscle in δ-SG KO mice. Collectively, these results confirm and extend understanding of a functional role for the DGC in the contractile properties of ASM and demonstrate that this results in altered lung function in vivo.

The N-terminal leucine-zipper motif in PTRF/cavin-1 is essential and sufficient for its caveolae-association

PTRF/cavin-1 is a protein of two lives. Its reported functions in ribosomal RNA synthesis and in caveolae formation happen in two different cellular locations: nucleus vs. plasma membrane. Here, we identified that the N-terminal leucine-zipper motif in PTRF/cavin-1 was essential for the protein to be associated with caveolae in plasma membrane. It could counteract the effect of nuclear localization sequence in the molecule (AA 235–251). Deletion of this leucine-zipper motif from PTRF/cavin-1 caused the mutant to be exclusively localized in nuclei. The fusion of this leucine-zipper motif with histone 2A, which is a nuclear protein, could induce the fusion protein to be exported from nucleus. Cell migration was greatly inhibited in PTRF/cavin-1−/− mouse embryonic fibroblasts (MEFs). The inhibited cell motility could only be rescued by exogenous cavin-1 but not the leucine-zipper motif deleted cavin-1 mutant. Plasma membrane dynamics is an important factor in cell motility control. Our results suggested that the membrane dynamics in cell migration is affected by caveolae associated PTRF/cavin-1.

TRPs in the kidney - location, location, location

TRPs in the kidney - location, location, location