Novel identification and modulation of the mechanosensitive Piezo1 channel in human myometrium.

Approximately 10% of US births deliver preterm before 37 weeks of completed gestation. Premature infants are at risk for life-long debilitating morbidities and death, and spontaneous preterm labour explains 50% of preterm births. In all cases existing treatments are ineffective, and none are FDA approved. The mechanisms that initiate preterm labour are not well understood but may result from dysfunctional regulation of quiescence mechanisms. Human pregnancy is accompanied by large increases in blood flow, and the uterus must enlarge by orders of magnitude to accommodate the growing fetus. This mechanical strain suggests that stretch-activated channels may constitute a mechanism to explain gestational quiescence. Here we identify for the first time that Piezo1, a mechanosensitive cation channel, is present in the uterine smooth muscle and microvascular endothelium of pregnant myometrium. Piezo is downregulated during preterm labour, and stimulation of myometrial Piezo1 in an organ bath with the agonist Yoda1 relaxes the tissue in a dose-dependent fashion. Further, stimulation of Piezo1 while inhibiting protein kinase A, AKT, or endothelial nitric oxide synthase mutes the negative inotropic effects of Piezo1 activation, intimating that actions on the myocyte and endothelial nitric oxide signalling contribute to Piezo1-mediated contractile dynamics. Taken together, these data highlight the importance of stretch-activated channels in pregnancy maintenance and parturition, and identify Piezo1 as a tocolytic target of interest. KEY POINTS: Spontaneous preterm labour is a serious obstetric dilemma without a known cause or effective treatments. Piezo1 is a stretch-activated channel important to muscle contractile dynamics. Piezo1 is present in the myometrium and is dysregulated in women who experience preterm labour. Activation of Piezo1 by the agonist Yoda1 relaxes the myometrium in a dose-dependent fashion, indicating that Piezo1 modulation may have therapeutic benefits to treat preterm labour.

Novel Tocolytic Strategy: Modulating Cx43 Activity by S-Nitrosation.

Currently available tocolytics are ineffective at significantly delaying preterm birth. This is due in part to our failure to better understand the mechanisms that drive spontaneous preterm labor (sPTL). Cyclic nucleotides are not the primary contributors to myometrial quiescence, but instead nitric oxide (NO)-mediated protein S-nitrosation (SNO) is integral to the relaxation of the tissue. Connexin-43 (Cx43), a myometrial "contractile-associated protein" that functions as either a gap junction channel or an hemichannel (HC), was the focus of this study. Protein analysis determined that Cx43 is downregulated in sPTL myometrium. Furthermore, Cx43 is S-nitrosated by NO, which correlates with an increase of phosphorylated Cx43 at serine 368 (Cx43-pS368 -gap junction inhibition) as well as an increase in the HC open-state probability (quiescence). Pharmacologic inhibition of Cx43 with 18ß-glycyrrhetinic acid (18ß-GA) exhibits a negative inotropic effect on the myometrium in a dose-dependent manner, as does administration of nebivolol, an NO synthase activator that increases total protein SNOs. When 18ß-GA and nebivolol were coadministered at their IC50 values, the effect on contractile dynamics was additive and all but eliminated contractions. The development of new tocolytics demands a better understanding of the underlying mechanisms of sPTL. Here it has been shown that 18ß-GA and nebivolol leverage dysregulated pathways in the myometrium, resulting in a novel approach for the treatment of sPTL. SIGNIFICANCE STATEMENT: Although there are many known causes of preterm labor (PTL), the mechanisms of "spontaneous" PTL (sPTL) remain obfuscated, which is why treating this condition is so challenging. Here we have identified that connexin-43 (Cx43), an important contractile-associated protein, is dysregulated in sPTL myometrium and that the pharmacologic inhibition of Cx43 and its S-nitrosation with 18ß-glycyrrhetinic acid and nebivolol, respectively, significantly blunts contraction in human myometrial tissue, presenting a novel approach to tocolysis that leverages maladjusted pathways in women who experience sPTL.

Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer.

Metastasis accounts for over 90% of cancer-related deaths, yet the mechanisms guiding this process remain unclear. Secreted nucleoside diphosphate kinase A and B (NDPK) support breast cancer metastasis. Proteomic evidence confirms their presence in breast cancer-derived extracellular vesicles (EVs). We investigated the role of EV-associated NDPK in modulating the host microenvironment in favor of pre-metastatic niche formation. We measured NDPK expression and activity in EVs isolated from triple-negative breast cancer (MDA-MB-231) and non-tumorigenic mammary epithelial (HME1) cells using flow cytometry, western blot, and ATP assay. We evaluated the effects of EV-associated NDPK on endothelial cell migration, vascular remodeling, and metastasis. We further assessed MDA-MB-231 EV-induced proteomic changes in support of pre-metastatic lung niche formation. NDPK-B expression and phosphotransferase activity were enriched in MDA-MB-231 EVs that promote vascular endothelial cell migration and disrupt monolayer integrity. MDA-MB-231 EV-treated mice demonstrate pulmonary vascular leakage and enhanced experimental lung metastasis, whereas treatment with an NDPK inhibitor or a P2Y1 purinoreceptor antagonist blunts these effects. We identified perturbations to the purinergic signaling pathway in experimental lungs, lending evidence to support a role for EV-associated NDPK-B in lung pre-metastatic niche formation and metastatic outgrowth. These studies prompt further evaluation of NDPK-mediated EV signaling using targeted genetic silencing approaches.

The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy.

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (•NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of •NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.

NF-κB/NKILA signaling modulates the anti-cancerous effects of EZH2 inhibition.

A wealth of evidence supports the broad therapeutic potential of NF-κB and EZH2 inhibitors as adjuvants for breast cancer treatment. We contribute to this knowledge by elucidating, for the first time, unique regulatory crosstalk between EZH2, NF-κB and the NF-κB interacting long non-coding RNA (NKILA). We define a novel signaling loop encompassing canonical and non-canonical actions of EZH2 on the regulation of NF-κB/NKILA homeostasis, with relevance to breast cancer treatment. We applied a respective silencing approach in non-transformed breast epithelial cells, triple negative MDA-MB-231 cells and hormone responsive MCF-7 cells, and measured changes in EZH2/NF-κB/NKILA levels to confirm their interdependence. We demonstrate cell line-specific fluctuations in these factors that functionally contribute to epithelial-to-mesenchymal transition (EMT) remodelling and cell fate response. EZH2 inhibition attenuates MDA-MB-231 cell motility and CDK4-mediated MCF-7 cell cycle regulation, while inducing global H3K27 methylation and an EMT phenotype in non-transformed cells. Notably, these events are mediated by a cell-context dependent gain or loss of NKILA and NF-κB. Depletion of NF-κB in non-transformed cells enhances their sensitivity to growth factor signaling and suggests a role for the host microenvironment milieu in regulating EZH2/NF-κB/NKILA homeostasis. Taken together, this knowledge critically informs the delivery and assessment of EZH2 inhibitors in breast cancer.

Hiding in Plain Sight: Nebivolol Exhibits Compelling Tocolytic Properties.

Preterm birth before 37 weeks of completed gestation results in numerous health consequences for the foetus. Preterm labour leads to preterm birth in over 50% of cases, and no FDA-approved treatment can prevent labour or help a foetus remain in the womb until term. Examination of nitric oxide mediated relaxation signaling in the uterine smooth muscle reveals a role for protein S-nitrosation. The recent discovery of upregulated S-nitrosoglutathione reductase (GSNOR) in spontaneously preterm labouring women has emphasized the need to explore the function of S-nitrosation regulation in the maintenance of uterine quiescence. Here we have examined the ability of nebivolol to relax uterine smooth muscle and tested recent claims that nebivolol is a GSNOR inhibitor. In uterine smooth muscle strips from both mouse and human, nebivolol relaxes oxytocin-induced contractions in a dose dependent manner. Our data indicates that nebivolol has no effect on GSNOR activity, nor does nebivolol inhibit thioredoxin reductase, two of the major protein denitrosylases. The ability of nebivolol to relax uterine smooth muscle is likely the combined effects of increased nitric oxide synthase activity and ß3-adregnegic stimulation.

Alternatively Spliced Human TREK-1 Variants Alter TREK-1 Channel Function and Localization.

TREK-1, an outward-rectifying potassium channel activated by stretch, is found in the myometrium of pregnant women. Decreased expression of TREK-1 near term suggests that TREK-1 may contribute to uterine quiescence during gestation. Five alternatively spliced TREK-1 variants were identified in the myometrium of mothers who delivered spontaneously preterm (<37 wk), leading to the hypothesis that these TREK-1 variants could interfere with TREK-1 function or expression. To investigate a potential role for these variants, immunofluorescence, cell surface assays, Western blots, and patch clamp were employed to study TREK-1 and TREK-1 variants expressed in HEK293T cells. The results of this study demonstrate that coexpression of TREK-1 with TREK-1 variants alters TREK-1 expression and suppresses channel function. Each variant affected TREK-1 in a disparate manner. In HEK293T cells coexpressing TREK-1 and each variant, TREK-1 membrane expression was diminished with compartmentalization inside the cell. When expressed alone, individual variants displayed channel properties that were significantly decreased compared to full-length TREK-1. In coexpression studies using patch clamp, basal TREK-1 currents were reduced by ∼64% (4.3 vs. 12.0 pA/pF) on average at 0 mV when coexpressed with each variant. TREK-1 currents that were activated by intracellular acidosis were reduced an average of ∼77% (21.4 vs. 94.5 pA/pF) at 0 mV when cells were transfected with TREK-1 and any one of the splice variants. These data correlate the presence of TREK-1 variants to reduced TREK-1 activity, suggesting a pathological role for TREK-1 variants in preterm labor.

The human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor.

Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and 1,000,000 die annually. Preterm labor results in preterm delivery in 50% of cases in the United States explaining 75% of fetal morbidity and mortality. There is no Food and Drug Administration-approved treatment to prevent preterm delivery. Nitric oxide-mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an nitric oxide donor, we identified 110 proteins that are S-nitrosated in 1 or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared with nonlaboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provide a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor.

TREK-1 currents in smooth muscle cells from pregnant human myometrium.

The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 µM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 µM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

Title: ß3 Adrenergic Receptor Signaling in the Human Myometrium.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. Although ß2 adrenergic agonists fail to provide adequate tocolysis, the expression of the ß3 adrenergic receptor in myometrium and its unique signaling suggest a role for ß3 agonist in the management of preterm labor. Western blot analysis showed that the ß3 adrenergic receptor expression increased in human pregnancy myometrium compared to nonpregnant tissues (p < 0.0001). There was no difference in ß3 adrenergic receptor expression throughout pregnancy (p > 0.05). The addition of the ß3 agonist mirabegron in the tissue bath relaxed oxytocin contracted myometrium with an EC50 of 41.5 µM. Relaxation was partially blocked by the addition of the eNOS blocker Nω-nitro-L-arginine, or the large conductance potassium channel blocker paxilline. Combination of Nω-nitro-L-arginine and paxilline prevented mirabegron-mediated relaxation. Imaging revealed that the ß3 adrenergic receptors are expressed by both myocyte and microvascular endothelial cells isolated from human myometrium. Nitric oxide production measured by 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate revealed that mirabegron stimulated nitric oxide production in myometrial endothelial cells. These data suggest that both endothelial and smooth muscle cells contribute to relaxation through disparate signaling pathways. Repurposing of approved medications tested in human myometrium as uterine tocolytics can advance prevention of preterm birth. These data argue that further examination of ß3 adrenergic receptor signaling in myometrium may reveal mirabegron as a useful tocolytic in combination tocolysis regimens.

ß3 Receptor Signaling in Pregnant Human Myometrium Suggests a Role for ß3 Agonists as Tocolytics.

Preterm labor leading to preterm birth is the leading cause of infant morbidity and mortality. At the present time, nothing can reliably halt labor once it begins. The knowledge that agonists of the ß2 adrenergic receptor relax airway smooth muscle and are effective in the treatment of asthma led to the notion that ß2 mimetics would prevent preterm birth by relaxing uterine smooth muscle. The activation of cAMP-dependent protein kinase by ß2 receptors is unable to provide meaningful tocolysis. The failure of ß2 agonists such as ritodrine and terbutaline to prevent preterm birth suggests that the regulation of uterine smooth muscle is disparate from that of airway. Other smooth muscle quiescent-mediating molecules, such as nitric oxide, relax vascular smooth muscle in a cGMP-protein kinase G-dependent manner; however, nitric oxide activation of protein kinase G fails to explain the relaxation of the myometrium to nitric oxide. Moreover, nitric oxide-mediated relaxation is blunted in preterm labor, and thus, for this reason and because of the fall in maternal blood pressure, nitric oxide cannot be employed as a tocolytic. The ß3 adrenergic receptor-mediated relaxation of the human myometrium is claimed to be cAMP-dependent protein kinase-dependent. This is scientifically displeasing given the failure of ß2 agonists as tocolytics and suggests a non-canonical signaling role for ß3AR in myometrium. The addition of the ß3 agonist mirabegron to pregnant human myometrial strips in the tissue bath relaxes oxytocin-induced contractions. Mirabegron stimulates nitric oxide production in myometrial microvascular endothelial cells, and the relaxation of uterine tissue in vitro is partially blocked by the addition of the endothelial nitric oxide synthase blocker Nω-Nitro-L-arginine. Recent data suggest that both endothelial and smooth muscle cells respond to ß3 stimulation and contribute to relaxation through disparate signaling pathways. The repurposing of approved medications such as mirabegron (Mybetriq™) tested in human myometrium as uterine tocolytics can advance the prevention of preterm birth.

ß3 adrenergic receptor activation modulates connexin 43 activity to relax human myometrium.

Preterm labor, delivery prior to 37 completed weeks of gestation, is the leading cause of infant morbidity and mortality. ß3 adrenergic receptor protein expression is increased in the myometrium during pregnancy, and the agonist, mirabegron, relaxes the myometrium making the ß3 adrenergic receptor a potential therapeutic target in PTL. ß3 adrenergic receptor has been shown to activate the tyrosine kinase, Src, which can down regulate connexin 43, a contractile associated protein which promotes the formation of gap junctions that create an electrical syncytium. We hypothesize that mirabegron downregulates connexin 43, imparting quiescence effects on the myometrium. Employing contractile studies, we demonstrate that Src is involved in the mirabegron-induced relaxation of contracting pregnant human myometrial tissue strips. Western blot analysis demonstrates that Src kinase expression is decreased in both preterm and term laboring myometrial tissue. Imaging revealed that mirabegron stimulation of the ß3 adrenergic receptor phosphorylates tyrosine at position Y265 on connexin 43 in pregnant human uterine myocytes. Western blot analysis and immunofluorescent imaging indicate that mirabegron decreases the expression of connexin 43 and mediates relaxation over a 24-h exposure period, suggesting that mirabegron has long lasting quiescent effects on the human myometrium. The relationship between the ß3 adrenergic receptor and down regulation of the contractile associated protein connexin 43 through activation of Src kinase suggests that mirabegron may be useful in combination tocolysis.

Uterine smooth muscle S-nitrosylproteome in pregnancy.

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not completely known. Nitric oxide relaxes uterine smooth muscle in a manner disparate from that for other smooth muscles because global elevation of cGMP after activation of soluble guanylyl cyclase does not relax the muscle. S-Nitrosylation, the covalent addition of an nitric oxide (NO) group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. This work is the first to describe the myometrial S-nitrosylproteome in both pregnant and nonpregnant tissue states. Using the guinea pig model, we show that specific sets of proteins involved in contraction and relaxation are S-nitrosylated in laboring and nonlaboring muscle and that many of these proteins are uniquely S-nitrosylated in only one state of the tissue. In particular, we show that S-nitrosylation of the intermediate filament protein desmin is significantly increased (5.7-fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8-fold, p < 0.005) that accompanies pregnancy. Elucidation of the myometrial S-nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction/relaxation.

Variants of stretch-activated two-pore potassium channel TREK-1 associated with preterm labor in humans.

Spontaneous preterm labor (PTL) is a uniquely human problem that results in preterm delivery of an underdeveloped fetus. The underlying cause remains elusive. The cost to societies in human suffering and treasure is enormous. The stretch-activated two pore potassium channel TREK-1 is up-regulated during gestation to term such that it may maintain uterine quiescence by hyperpolarizing the smooth muscle cell membrane. We have hypothesized that the human TREK-1 channel is involved in myometrial relaxation during pregnancy and that splice variants of the TREK-1 channel expressed in preterm myometrium are associated with preterm delivery by interaction with full-length TREK-1. We detected three wild-type human TREK-1 transcript isoforms in nonpregnant and pregnant human myometrium. Using RT-PCR, we identified five unique TREK-1 splice variants in myometrium from women in PTL. These myometrial TREK-1 variants lack either the pore or the transmembrane domains or both. In transiently transfected HEK293T cells, wild-type TREK-1 was predominantly expressed at the plasma membrane. However, individual splice variants were expressed uniformly throughout the cell. Wild-type TREK-1 was localized at the plasma membrane and cytoplasm close to the plasma membrane when coexpressed with each splice variant. Co-immunoprecipitation of FLAG epitope-tagged TREK-1 and six-His epitope-tagged splice variants using Ni bead columns successfully pulled down wild-type TREK-1. These results suggest that each of four TREK-1 splice variants interacts with full-length wild-type TREK-1 and that in vivo, such interactions may contribute to a PTL phenotype.

Evolution of Medical Approaches and Prominent Therapies in Breast Cancer.

An examination of the origins of medical approaches to breast cancer marks this disease as one of the most difficult to manage. As the early identification, diagnosis and treatment of breast cancer evolve, we will move to a time when each patient and their cancer can be assessed to determine unique patient-specific (personalized) approaches to therapy. Humans have attempted to manage breast cancer for millennia. Even today, the disease claims thousands of lives each year. In light of the increasingly sophisticated understanding of cancer diagnosis and treatment, together with our ultimate failure to offer a cure in the most difficult cases, it is instructive to reflect on the beginnings of our understanding.

A role of stretch-activated potassium currents in the regulation of uterine smooth muscle contraction.

Rates of premature birth are alarming and threaten societies and healthcare systems worldwide. Premature labor results in premature birth in over 50% of cases. Preterm birth accounts for three-quarters of infant morbidity and mortality. Children that survive birth before 34 weeks gestation often face life-long disability. Current treatments for preterm labor are wanting. No treatment has been found to be generally effective and none are systematically evaluated beyond 48 h. New approaches to the treatment of preterm labor are desperately needed. Recent studies from our laboratory suggest that the uterine muscle is a unique compartment with regulation of uterine relaxation unlike that of other smooth muscles. Here we discuss recent evidence that the mechanically activated 2-pore potassium channel, TREK-1, may contribute to contraction-relaxation signaling in uterine smooth muscle and that TREK-1 gene variants associated with human labor and preterm labor may lead to a better understanding of preterm labor and its possible prevention.

Agonist-specific compartmentation of cGMP action in myometrium.

Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin-particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.

Mechanical strain induced phospho-proteomic signaling in uterine smooth muscle cells.

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

S-Nitrosoglutathione Reductase Underlies the Dysfunctional Relaxation to Nitric Oxide in Preterm Labor.

Tocolytics show limited efficacy to prevent preterm delivery. In uterine smooth muscle cGMP accumulation following addition of nitric oxide (NO) has little effect on relaxation suggesting a role for protein S-nitrosation. In human myometrial tissues from women in labor at term (TL), or spontaneously in labor preterm (sPTL), direct stimulation of soluble guanylyl cyclase (sGC) fails to relax myometrium, while the same treatment relaxes vascular smooth muscle completely. Unlike term myometrium, effects of NO are not only blunted in sPTL, but global protein S-nitrosation is also diminished, suggesting a dysfunctional response to NO-mediated protein S-nitrosation. Examination of the enzymatic regulator of endogenous S-nitrosoglutathione availability, S-nitrosoglutathione reductase, reveals increased expression of the reductase in preterm myometrium associated with decreased total protein S-nitrosation. Blockade of S-nitrosoglutathione reductase relaxes sPTL tissue. Addition of NO donor to the actin motility assay attenuates force. Failure of sGC activation to mediate relaxation in sPTL tissues, together with the ability of NO to relax TL, but not sPTL myometrium, suggests a unique pathway for NO-mediated relaxation in myometrium. Our results suggest that examining the action of S-nitrosation on critical contraction associated proteins central to the regulation of uterine smooth muscle contraction can reveal new tocolytic targets.

Functional compartmentation of endothelial P2Y receptor signaling.

The presence of multiple receptors for disparate nucleotides on endothelial cells makes it unclear how the endothelium differentiates among these signals. We propose that endothelial P2Y receptors are organized into cholesterol-rich signaling domains, such as caveolae and respond to nucleotide agonists by mobilizing intracellular calcium. Treatment of endothelial cells with 5 mmol/L beta-methyl-cyclodextrin prevents calcium release in response to the nucleotide receptor agonists 2-methylthio-ATP, ATP, ADP, and UTP, but not the kinin receptor agonist bradykinin, suggesting that depletion of membrane cholesterol disrupts signaling at P2Y receptors and that bradykinin receptors are not prelocalized to cholesterol microdomains in these cells. Direct measurement of cholesterol content after beta-methyl-cyclodextrin treatment of aortic rings reveals a concentration-dependent depletion of cholesterol that parallels functional antagonism of P2Y-mediated relaxation. Nucleotide- and bradykinin-mediated relaxation is disrupted by 5 to 15 mmol/L beta -methyl-cyclodextrin treatment or 1 to 10 microg/mL filipin III in a concentration-dependent fashion. Norepinephrine contracted aorta treated with A23187 relaxes in an endothelium-dependent fashion despite depletion of 84% of membrane-extractable cholesterol. These data indicate that in the basal state, P2Y receptors but not the kinin receptor may be compartmented to cholesterol-dependent signaling domains in guinea pig endothelium and that cholesterol-rich microdomains in these cells can respond to intracellular calcium in an agonist-specific manner. We suggest that the functional organization of cholesterol-rich signaling microdomains allows agonist-specific responses to increases in intracellular calcium and that this property may be a general phenomenon that permits cells to respond disparately to agonists that may signal through common calcium release pathways.