Prolylcarboxypeptidase Alleviates Hypertensive Cardiac Remodeling by Regulating Myocardial Tissue Angiotensin II.

Background Prolonged activation of angiotensin II is the main mediator that contributes to the development of heart diseases, so converting angiotensin II into angiotensin 1-7 has emerged as a new strategy to attenuate detrimental effects of angiotensin II. Prolylcarboxypeptidase is a lysosomal pro-X carboxypeptidase that is able to cleave angiotensin II at a preferential acidic pH optimum. However, insufficient attention has been given to the cardioprotective functions of prolylcarboxylpeptidase. Methods and Results We established a CRISPR/CRISPR-associated protein 9-mediated global prolylcarboxylpeptidase-knockout and adeno-associated virus serotype 9-mediated cardiac prolylcarboxylpeptidase overexpression mouse models, which were challenged with the angiotensin II infusion (2 mg/kg per day) for 4 weeks, aiming to investigate the cardioprotective effect of prolylcarboxylpeptidase against hypertensive cardiac hypertrophy. Prolylcarboxylpeptidase expression was upregulated after 2 weeks of angiotensin II infusion and then became downregulated afterward in wild-type mouse myocardium, suggesting its compensatory function against angiotensin II stress. Moreover, angiotensin II-treated prolylcarboxylpeptidase-knockout mice showed aggravated cardiac remodeling and dampened cardiac contractility independent of hypertension. We also found that prolylcarboxylpeptidase localizes in cardiomyocyte lysosomes, and loss of prolylcarboxylpeptidase led to excessive angiotensin II levels in myocardial tissue. Further screening demonstrated that hypertrophic prolylcarboxylpeptidase-knockout hearts showed upregulated extracellular signal-regulated kinases 1/2 and downregulated protein kinase B activities. Importantly, adeno-associated virus serotype 9-mediated restoration of prolylcarboxylpeptidase expression in prolylcarboxylpeptidase-knockout hearts alleviated angiotensin II-induced hypertrophy, fibrosis, and cell death. Interestingly, the combination of adeno-associated virus serotype 9-mediated prolylcarboxylpeptidase overexpression and an antihypertensive drug, losartan, likely conferred more effective protection than a single treatment protocol to mitigate angiotensin II-induced cardiac dysfunction. Conclusions Our data demonstrate that prolylcarboxylpeptidase protects the heart from angiotensin II-induced hypertrophic remodeling by controlling myocardial angiotensin II levels.

The R-Domain: Identification of an N-terminal Region of the α2δ-1 Subunit Which is Necessary and Sufficient for its Effects on Cav2.2 Calcium Currents.

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming α1 and modulatory ß and α2δ subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the α2δ subunit remains lacking. Using molecular biology and electrophysiological approaches we have conducted the first systematic mapping of α2δ subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat α2δ-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N- to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of α2δ-1 - we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electrophysiological and trafficking effects of α2δ-1 on Cav. Structural algorithms predict that Rd is conserved across all four α2δ subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the α1 subunit and may provide a target for novel Cav therapeutics.

Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation.

Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na(+) channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer.

Functional expression of the voltage-gated Na⁺-channel Nav1.7 is necessary for EGF-mediated invasion in human non-small cell lung cancer cells.

Various ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion and metastasis. Expression of functional voltage-gated Na(+) channels (Nav) is implicated in the metastatic potential of breast, prostate, lung and colon cancer cells. However, the cellular mechanisms that regulate Nav expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and Nav in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the α subunit Nav1.7 promotes invasion in H460 NSCLC cells. Inhibition of Nav1.7 activity (using tetrodotoxin) or expression (by using small interfering RNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Nav, whereas exogenous overexpression of the Nav1.7 α subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhances proliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Nav1.7 is necessary for invasive behaviour in these cells. Examination of Nav1.7 expression at mRNA, protein and functional levels further reveals that EGF/EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion. Immunohistochemistry of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significant potential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.

Association of physical activity and sedentary behavior with biological markers among U.S. pregnant women.

BACKGROUND: To examine the association between objectively measured light-intensity and moderate-to-vigorous-intensity physical activity (MVPA), sedentary behaviors, and biological markers in a national sample of U.S. pregnant women, as few studies have examined these relationships among this population. METHODS: The sample of noninstitutionalized U.S. civilians was selected by a complex, multistage probability design. Data from the 2003-2006 National Health and Examination Survey were used. Two hundred six pregnant women were included in the data analysis. Physical activity and sedentary data were objectively measured via accelerometry (ActiGraph 7164). Biomarker data was obtained in the mobile examination center from urine, blood samples, blood pressure, and anthropometric measurements. Urine and blood samples were obtained to determine pregnancy status, C-reactive protein (CRP), high-density lipoprotein (HDL) cholesterol, total cholesterol, and cotinine as well as fasting glucose, fasting triglycerides, and fasting low-density lipoprotein (LDL) cholesterol data. Multivariable regression was employed to examine the association between physical activity, sedentary behavior, and biomarker levels. RESULTS: There was a positive association between sedentary behavior and CRP levels (beta coefficient [b]=0.001, p=0.02) and LDL cholesterol (b=0.12, p=0.02). There was an inverse association between light-intensity physical activity and CRP (b=-0.003; p=0.008) and diastolic blood pressure (b=-0.03; p=0.02), with those engaging in higher levels of MVPA having higher HDL cholesterol (b=6.7; p=0.01). CONCLUSION: Physical activity and sedentary behavior were favorably associated with various biomarkers among pregnant women, suggesting that healthcare providers should encourage pregnant women to participate in safe forms of physical activity behaviors while also reducing their amount of time spent in sedentary behaviors.

Physical activity and depression symptoms among pregnant women from the National Health and Nutrition Examination Survey 2005-2006.

OBJECTIVE: To examine the association between objectively measured physical activity and depression symptoms among a nationally representative sample of pregnant women to provide a more accurate understanding of the relationship between physical activity and depression symptoms. DESIGN: We employed a cross-sectional study design. SETTING: Data from the National Health and Nutrition Examination Survey 2005-2006 were used for this study. METHODS: One-hundred and forty-one pregnant women wore an ActiGraph accelerometer for 7 days and completed the Patient Health Questionnaire-9 to assess depression status. RESULTS: More than 19% of the participants experienced some depression symptoms, and compared to their counterparts not having depression symptoms, they were less physically active. CONCLUSION: An inverse association was found between physical activity and depression symptoms among pregnant women. When feasible, nurses are encouraged to help facilitate physical activity among pregnant women, assuming an uncomplicated pregnancy.

Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif.

Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory ß and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α(2)δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein--PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α(2)δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α(2)δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α(2)δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.

Formation of N-type (Cav2.2) voltage-gated calcium channel membrane microdomains: Lipid raft association and clustering.

Voltage-gated calcium channels (Ca(v)s) comprise a pore-forming α1 with auxiliary α2δ and ß subunits which modulate Ca(v) function and surface expression. Ca(v)α1 and α2δ are present in signalling complexes termed lipid rafts but it is unclear whether α2δ is obligatory for targeting Ca(v)s to rafts or to what extent this influences cell surface organisation of Ca(v)s. Here, we have used imaging, biochemistry and electrophysiology to determine localisation and raft-partitioning of WT and functionally active HA-epitope tagged α2δ-1 and Ca(v)2.2 subunits expressed in COS-7 cells. We show that α2δ-1 not only partitions into lipid rafts itself but also mediates raft-partitioning of Ca(v)2.2/ß(1b) complexes. Ca(v)α2δ-1, Ca(v)2.2/ß(1b) and Ca(v)2.2/ß(1b)/α2δ-1 complexes are all organised into cell surface clusters although only in the presence of α2δ-1 do they co-localise with raft markers, caveolin and flotillin. Such clusters persist in the presence of 3-methyl-ß-cyclodextrin even though the raft markers disperse. However, clustering is profoundly sensitive to disruption of the actin-based cytoskeleton by cytochalasin-D. We conclude that α2δ-1, and likely other α2δ subunits, is necessary and sufficient for targeting Ca(v)s to lipid rafts. However, formation of clusters supporting "hotspots" of Ca(v) activity requires aggregation of macromolecular complexes containing raft components, stabilised by interactions with the cytoskeleton.

The presence of Ca2+ channel beta subunit is required for mitogen-activated protein kinase (MAPK)-dependent modulation of alpha1B Ca2+ channels in COS-7 cells.

In rat sensory neurones, voltage-dependent calcium channels (VDCCs), including the N-type, are tonically up-regulated via Ras/mitogen-activated protein kinase (MAPK) signalling. To determine whether VDCC beta subunit is involved in this process, the role of the four neuronal betas (beta1b, beta2a, beta3, beta4) in MAPK-dependent modulation of alpha1B (Ca(v)2.2, N-type) Ca(2+) channels has been examined in COS-7 cells. MAPK is exclusively activated by MAPK kinase (MEK), and here, acute application of a MEK-specific inhibitor UO126, significantly inhibited peak alpha1B Ca2+ channel current (I(max)) within a period of 5-10 min, regardless of which beta subunit was co-expressed (25-50 %, P < 0.01). With beta2a however, the percentage inhibition of I(max) was less than that observed with any other beta (ANOVA: F(3,34) = 6.48, P < 0.01). UO126 also caused a hyperpolarising shift (6 +/- 1 mV, P < 0.001) in the voltage dependence of beta2a current activation, such that inhibition occurred only at depolarised potentials (> +5 mV) whereas at more negative potentials the current amplitude was enhanced. A marked change in beta2a current kinetics, perceived either as decreased activation or increased inactivation, was also associated with UO126 application. A similar effect of UO126 on beta4 current kinetics was also observed. The beta2a-specific effects of UO126 on current inhibition and voltage dependence of activation were abolished when alpha1B was co-expressed with de-palmitoylated beta2a(C3,4S), in which amino terminal cysteines 3 and 4 had been mutated to serines. In the absence of beta subunit, UO126 had no effect on alpha1B Ca2+ channel current. Together, these data suggest an absolute requirement for beta in MAPK-dependent modulation of these channels. Since beta subunits vary both in their temporal expression and localisation within neurones, beta subunit-dependent modulation of N-type Ca2+ channels via MAPK could provide an important new mechanism by which to fine-tune neurotransmitter release.