The scaffold protein IQGAP1 is crucial for extravasation and metastasis.

IQGAP1 is a scaffold protein involved in a range of cellular activities, including migration, invasion, adhesion and proliferation. It is also oncogenic in a variety of cancers, promoting primary tumor growth and invasiveness. However, the role of IQGAP1 in tumor progression and metastasis remains unclear. In this study, we use both knockdown and knockout of IQGAP1 to investigate its role in the metastatic cascade of both melanoma and breast cancer cells in vivo. We find that reduction of IQGAP1 expression decreases the formation of both spontaneous and experimental metastases, without limiting primary or metastatic tumor growth. Furthermore, IQGAP1 knockout significantly inhibits extravasation of tumor cells from circulation, possibly involving invadopodial function. By expressing mutant forms of IQGAP1 in a knockout context, we also determine that IQGAP1's pro-metastatic functions are dependent on multiple domains and functions. These data demonstrate that IQGAP1 is crucial for metastasis in vivo through regulation of extravasation and suggest that it may represent a valid therapeutic target for inhibiting metastasis.

Large conductance Ca2+-activated K+ channels modulate uterine α1-adrenergic sensitivity in ovine pregnancy.

The uteroplacental vasculature is refractory to α-adrenergic stimulation, and large conductance Ca(2+)-activated K(+) channels (BK(Ca)) may contribute. We examined the effects of uterine artery (UA) BK(Ca) inhibition with tetraethylammonium (TEA) on hemodynamic responses to phenylephrine (PE) at 101 to 117 days and 135 to 147 days of ovine gestation, obtaining dose responses for mean arterial pressure (MAP), heart rate (HR), and uteroplacental blood flow (UPBF) and vascular resistance (UPVR) before and during UA TEA infusions. The UA α(1)-adrenergic receptors (α1-ARs) were assessed. The PE increased MAP and UPVR and decreased HR and UPBF dose dependently at both gestations (P < .001, analysis of variance). The %▵MAP was less at 135 to 147 days before and during TEA infusions (P ≤ .008); however, responses during TEA were greater (P ≤ .002). The PE increased %▵UPVR>>%▵MAP, thus %▵UPBF fell. The TEA enhanced PE-mediated increases in %▵UPVR at 135 to 147 days (P ≤ .03). The UA α(1)-AR expression was unchanged in pregnancy. Uterine vascular responses to PE exceed systemic vascular responses throughout pregnancy and are attenuated by BK(Ca) activation, suggesting BK(Ca) protect UPBF.

Differential sensitivity to angiotensin II and norepinephrine in human uterine arteries.

BACKGROUND: During pregnancy, uteroplacental responses to norepinephrine (NE) exceed systemic responses. In contrast, uteroplacental responses to angiotensin II (ANG II) are less than systemic. The explanation for these differences in uteroplacental sensitivity remain unclear but may reflect type 2 ANG II receptor (AT(2)R) predominance in uterine artery (UA) vascular smooth muscle (VSM). OBJECTIVE: The objective of the study was to examine VSM sensitivity to KCl, NE, and ANG II in UA from nonpregnant (NP) and pregnant (P) women and determine VSM ANG II receptor subtype expression. METHODS: Responses to KCl, NE, and ANG II were examined in endothelium-denuded UA rings from NP (n = 28) and P (n = 13; 34-40 wk gestation) women, and ANG II receptor subtype, α(1)-receptor and contractile proteins were measured. RESULTS: KCl and NE dose dependently contracted UA (P < 0.001), P exceeding NP 2-fold or greater; but α(1)-receptor expression was unchanged. ANG II did not elicit dose effects in NP or P UA; however, P responses exceeded NP approximately 2-fold (P < 0.001) and were approximately 2.5-fold less than NE (P < 0.001). AT(2)R and AT(1)R expression were similar (P > 0.1) in VSM from NP and term P women. AT(1)R blockade abolished ANG II contractions (P < 0.001); AT(2)R blockade did not enhance ANG II sensitivity in UA with or without endothelium. Actin contents increased approximately 2-fold in term UA. CONCLUSIONS: Sensitivity to α-stimulation exceeds ANG II in NP and P UA, explaining the differential uteroplacental sensitivity in pregnancy. Because AT(2)R predominate in UA VSM throughout reproduction, this contributes to the inherent refractoriness to ANG II in the uterine vasculature. The increase in UA contractile proteins at term P suggests remodeling, explaining the enhanced contractility seen.

Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature.

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT(2) receptors (AT(2)R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant (n = 16) and term pregnant (n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner (P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM (P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content (P ≤ 0.01). UVSM AT(1)R and AT(2)R expression was similar throughout the UVB and unchanged during pregnancy (P > 0.1). AT(1)R inhibition blocked ANG II-mediated contractions; AT(2)R blockade, however, did not enhance contractions. AT(2)R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

Regulation of the cGMP-cPKG pathway and large-conductance Ca2+-activated K+ channels in uterine arteries during the ovine ovarian cycle.

The follicular phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular PGF(2alpha), CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The pore-forming alpha- and regulatory beta-subunits that constitute the BK(Ca), soluble guanylyl cyclase (sGC), and cGMP-dependent protein kinase G (cPKG) isoforms (cPKG(1alpha) and cPKG(1beta)) were measured by Western analysis and cGMP levels by RIA. BK(Ca) subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in cPKG(1alpha) (P < 0.05). sGC, cPKG(1beta), and the BK(Ca) alpha-subunit were unchanged. Notably, expression of beta(1)- and beta(2)-regulatory subunits rose 51 and 79% (P

Pregnancy modifies the large conductance Ca2+-activated K+ channel and cGMP-dependent signaling pathway in uterine vascular smooth muscle.

Regulation of uteroplacental blood flow (UPBF) during pregnancy remains unclear. Large conductance, Ca(2+)-activated K(+) channels (BK(Ca)), consisting of alpha- and regulatory beta-subunits, are expressed in uterine vascular smooth muscle (UVSM) and contribute to the maintenance of UPBF in the last third of ovine pregnancy, but their expression pattern and activation pathways are unclear. We examined BK(Ca) subunit expression, the cGMP-dependent signaling pathway, and the functional role of BK(Ca) in uterine arteries (UA) from nonpregnant (n = 7), pregnant (n = 38; 56-145 days gestation; term, approximately 150 days), and postpartum (n = 15; 2-56 days) sheep. The alpha-subunit protein switched from 83-87 and 105 kDa forms in nonpregnant UVSM to 100 kDa throughout pregnancy, reversal occurring >30 days postpartum. The 39-kDa beta(1)-subunit was the primary regulatory subunit. Levels of 100-kDa alpha-subunit rose approximately 70% during placentation (P < 0.05) and were unchanged in the last two-thirds of pregnancy; in contrast, beta(1)-protein rose throughout pregnancy (R(2) = 0.996; P < 0.001; n = 13), increasing 50% during placentation and approximately twofold in the remainder of gestation. Although UVSM soluble guanylyl cyclase was unchanged, cGMP and protein kinase G(1alpha) increased (P < 0.02), paralleling the rise and fall in beta(1)-protein during pregnancy and the puerperium. BK(Ca) inhibition not only decreased UA nitric oxide (NO)-induced relaxation but also enhanced alpha-agonist-induced vasoconstriction. UVSM BK(Ca) modify relaxation-contraction responses in the last two-thirds of ovine pregnancy, and this is associated with alterations in alpha-subunit composition, alpha:beta(1)-subunit stoichiometry, and upregulation of the cGMP-dependent pathway, suggesting that BK(Ca) activation via NO-cGMP and beta(1) augmentation may contribute to the regulation of UPBF.

Large conductance Ca2+-activated K+ channels contribute to vascular function in nonpregnant human uterine arteries.

Large conductance K( +) channels (BK(Ca)) are expressed in uterine artery (UA) smooth muscle from nonpregnant and pregnant sheep and contribute to the regulation of basal vascular tone and responses to estrogen and vasoconstrictors. To determine if BK(Ca) are expressed in women and contribute to UA function, we collected UA from nonpregnant women (n = 31) at elective hysterectomy and analyzed for subunit protein, localization with immunohistochemistry, and function using endothelium-denuded rings. UA expresses BK(Ca) alpha -, beta1- and beta2-subunit protein. KCl and phenylephrine (PE, an alpha(1)-agonist) caused dose-dependent vasoconstriction (P < .001), and UA precontracted with PE dose-dependently relaxed with sodium nitroprusside (SNP; P < .001).Tetraethylammonium chloride (TEA, 0.2-1.0 mM), a BK(Ca) inhibitor, dose-dependently increased resting tone (P = .004; 28% +/- 5.3% with 1.0 mM), enhanced PE-induced (10(-)(6) M) vasoconstriction (P < .04), and attenuated SNP-induced relaxation at 1.0 mM (P = .02). BK( Ca) are expressed in human UA and modulate vascular function by attenuating vasoconstrictor responses and contributing to nitric oxide-induced vasorelaxation.

Meconium increases type 1 angiotensin II receptor expression and alveolar cell death.

The pulmonary renin-angiotensin system (RAS) contributes to inflammation and epithelial apoptosis in meconium aspiration. It is unclear if both angiotensin II receptors (ATR) contribute, where they are expressed and if meconium modifies subtype expression. We examined ATR subtypes in 2 wk rabbit pup lungs before and after meconium exposure and with and without captopril pretreatment or type 1 receptor (AT1R) inhibition with losartan, determining expression and cellular localization with immunoblots, RT-PCR and immunohistochemistry, respectively. Responses of cultured rat alveolar type II pneumocytes were also examined. Type 2 ATR were undetected in newborn lung before and after meconium instillation. AT1R were expressed in pulmonary vascular and bronchial smooth muscle and alveolar and bronchial epithelium. Meconium increased total lung AT1R protein approximately 3-fold (p = 0.006), mRNA 29% (p = 0.006) and immunostaining in bronchial and alveolar epithelium and smooth muscle, which were unaffected by captopril and losartan. Meconium also increased AT1R expression >3-fold in cultured type II pneumocytes and caused concentration-dependent cell death inhibited by losartan. Meconium increases AT1R expression in newborn rabbit lung and cultured type II pneumocytes and induces AT1R-mediated cell death. The pulmonary RAS contributes to the pathogenesis of meconium aspiration through increased receptor expression.

Vascular development in early ovine gestation: carotid smooth muscle function, phenotype, and biochemical markers.

Vascular smooth muscle (VSM) maturation is developmentally regulated and differs between vascular beds. The maturation and contribution of VSM function to tissue blood flow and blood pressure regulation during early gestation are unknown. The carotid artery (CA) contributes to fetal cerebral blood flow regulation and well being. We studied CA VSM contractility, protein contents, and phenotype beginning in the midthird of ovine development. CAs were collected from early (88-101 day of gestation) and late (138-150 day; term = day 150) fetal (n = 14), newborn (6-8 day old; n = 7), and adult (n = 5) sheep to measure forces in endothelium-denuded rings with KCl, phenylephrine, and ANG II; changes in cellular proteins, including total and soluble protein, actin and myosin, myosin heavy chain isoforms (MHC), filamin, and proliferating cell nuclear antigen; and vascular remodeling. KCl and phenylephrine elicited age- and dose-dependent contraction responses (P < 0.001) at all ages except early fetal, which were unresponsive. In contrast, ANG II elicited dose responses only in adults, with contractility increasing greater than fivefold vs. that shown in fetal or neonatal animals (P < 0.001). Increased contractility paralleled age-dependent increases (P < 0.01) in soluble protein, actin and myosin, filamin, adult smooth muscle MHC-2 (SM2) and medial wall thickness and reciprocal decreases (P < 0.001) in nonmuscle MHC-B, proliferating cell nuclear antigen and medial cellular density. VSM nonreceptor- and receptor-mediated contractions are absent or markedly attenuated in midgestation and increase age dependently, paralleling the transition from synthetic to contractile VSM phenotype and, in the case of ANG II, paralleling the switch to the AT(1) receptor. The mechanisms regulating VSM maturation and thus blood pressure and tissue perfusion in early development remain to be determined.

Distribution of co-activators CBP and p300 during mouse oocyte and embryo development.

cAMP response element binding protein (CREB)-binding protein (CBP) and p300 are two structurally related transcriptional co-activators that activate expression of many eukaryotic genes. Current dogma would suggest that these transcriptional co-activators have similar mechanisms of transcription regulation. Studies of CBP or p300 homozygotic mouse mutants indicate that normal embryogenesis requires the existence of both factors. However, whether this is indicative of a dosage effect of these two proteins, or whether these proteins play different roles in mouse embryo development is not clear. Here we demonstrated that both factors are first found in the cytoplasm of oocytes within primordial follicles, and that they enter into the oocyte nucleus at different stages of oocyte growth, suggesting that they may play different roles in gene expression during oocyte growth and development. Consistent with this model, in the pre-implantation mouse embryos, from the two-cell stage to the blastocyst stage, the localizations of CBP and p300 are different, at times opposite, indicating that CBP and p300 also have different functions in early mouse embryogenesis.

Estrogen regulates {beta}1-subunit expression in Ca(2+)-activated K(+) channels in arteries from reproductive tissues.

Daily estradiol-17beta (E(2)beta) increases basal uterine blood flow (UBF) and enhances acute E(2)beta-mediated increases in UBF in ovariectomized nonpregnant ewes. The acute E(2)beta-mediated rise in UBF involves vascular smooth muscle (VSM) large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). BK(Ca) consist of pore-forming alpha-subunits and regulatory beta(1)-subunits that modulate channel function and E(2)beta responsiveness. It is unclear whether E(2)beta also alters subunit expression and thus channel density and/or function, thereby contributing to the rise in basal UBF and enhanced UBF responses that follow daily E(2)beta. Therefore, we examined BK(Ca) subunit expression by using reverse transcription-PCR and immunoblot analysis of arterial VSM from reproductive and nonreproductive tissues and myometrium from ovariectomized nonpregnant ewes after daily E(2)beta (1 microg/kg iv) or vehicle without or with acute E(2)beta (1 microg/kg). Tissue distribution was determined by immunohistochemistry. Acute E(2)beta did not alter alpha- or beta(1)-subunit expression in any tissue (P > 0.1). Daily E(2)beta also did not affect alpha-subunit mRNA or protein in any tissue (P > 0.1) or mesenteric arterial VSM beta(1)-subunit. However, daily E(2)beta increased uterine and mammary arterial VSM beta(1)-subunit mRNA by 32% and 83% (P < 0.05), uterine VSM protein by 30%, and myometrial beta(1)-subunit mRNA and protein by 74% (P < or = 0.005). Immunostaining of uterine arteries, myometrium, and intramyometrial arteries paralleled immunoblot analyses for both subunits. Although BK(Ca) density is unaffected by daily and acute E(2)beta, daily E(2)beta increases beta(1)-subunit in proximal and distal uterine arterial VSM. Thus prolonged E(2)beta exposure may alter BK(Ca) function, estrogen responsiveness, and basal vascular tone and reactivity in reproductive arteries by modifying alpha:beta(1) stoichiometry.

Vessel-specific regulation of angiotensin II receptor subtypes during ovine development.

Umbilical and systemic responses to angiotensin II differ in term fetal sheep, and peripheral vascular responses are attenuated or absent before and after birth. These observations may reflect developmental differences in angiotensin II receptor (AT) subtypes in vascular smooth muscle (VSM). Studies of AT subtype ontogeny and regulation are generally limited to the aorta, which may not be extrapolated to other arteries, and neither is completely described during ovine development. We therefore characterized VSM AT subtype expression and regulation throughout an extended period of development in umbilical and carotid artery and aorta from fetal (85-146 d gestation), postnatal (5-23 d), and adult sheep, measuring AT(1) and AT(2) mRNA and protein and performing immunohistochemistry. Parallel increases in umbilical AT(1) mRNA and protein began early in gestation and continued to term, and although AT(2) mRNA was unchanged, protein levels decreased >90% at term. Fetal carotid AT(1) mRNA was <40% of adult values and unchanged before birth; however, AT(1) protein rose >2-fold at term. After birth, AT(1) mRNA increased to 85% of adult values and was associated with another 2-fold rise in protein. In contrast, carotid AT(2) mRNA and protein fell in parallel throughout development and were barely detectable in the newborn and the adult. Immunostaining was consistent with observations in both arteries. A third pattern occurred in aortic VSM. The ontogeny of AT subtype expression and regulation is vessel specific, with changes in umbilical VSM beginning very early in development. Although the mechanisms that regulate mRNA and protein expression are unclear, these changes parallel differences in VSM maturation and function and local blood flow.

Endogenous regulators of protein phosphatase-1 during mouse oocyte development and meiosis.

Reversible phosphorylation, involving protein kinases and phosphatases (PP), is important in regulating oocyte meiosis. Okadaic acid (OA) inhibition of PP1 and/or PP2A stimulates oocyte germinal vesicle breakdown (GVB). In oocytes, PP1 is localized in the cytoplasm and nucleus, yet endogenous regulation of oocyte PP1 has not been investigated. The objectives of the study were to identify intra-oocyte mechanisms regulating PP1 during acquisition of OA-sensitive meiotic competence and meiotic resumption. Immunohistochemical studies revealed that GVB-incompetent oocytes contained equivalent cytoplasmic and nuclear PP1. Upon development of OA-sensitive meiotic competence, PP1 displayed differential intracellular localization with significantly greater nuclear staining with distinct nucleolar rimming compared with cytoplasmic staining. Germinal vesicle-intact oocytes contained neither nuclear inhibitor of PP1, nor PP1 cytoplasmic inhibitor-1 transcripts or proteins. Reverse transcription-PCR with PP1 cytoplasmic inhibitor-2 (I2) primers and oocyte RNA amplified a predicted 330-bp product with the identical sequence to mouse liver I2. Oocytes contained a heat-stable PP1 inhibitor with biochemical properties of I2. Phosphorylation of PP1 at Thr320 by cyclin dependent kinase-1 (CDK1) causes PP1 inactivation. Germinal vesicle-intact oocytes did not contain phospho-Thr320-PP1. Upon GVB, PP1 became phosphorylated at Thr320 and this phosphorylation did not occur if GVB was blocked with the CDK1 inhibitor, roscovitine (ROSC). Inhibition of oocyte GVB with ROSC was reversible and coincided with PP1 phosphorylation at Thr320. Increased oocyte staining of nuclear PP1 compared with cytoplasmic staining at a chronological stage when oocytes gain meiotic competence, and phosphorylation and inhibition of PP1 by CDK1 at or around GVB appear to be important mechanisms in regulating oocyte PP1 activity and meiosis. In addition, these studies provide further support for PP1 being the OA-sensitive PP important in the regulation of the acquisition of meiotic competence, nuclear events during meiotic arrest, and GVB.

Glycogen synthase kinase-3 regulates mouse oocyte homologue segregation.

Intracellular regulation of oocyte meiosis is not completely understood. However, reversible phosphorylation, which involves serine/threonine protein kinases and phosphatases (PP), is an important mediator. Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase. Currently no reports exist on presence or function of GSK-3 in mammalian oocytes. The aim of this study was to determine GSK-3 presence/absence, transcript and protein expression, intracellular protein distribution, and to investigate the functional importance of GSK-3 in mouse oocyte meiosis. Germinal vesicle-intact (GVI) oocytes contained both GSK-3 transcript and protein. Although GSK-3 beta-isoform is the only transcript identifiable in GVI oocytes, both alpha- and beta-isoforms were recognized by Western blot analysis. In growing, meiotic-incompetent oocytes GSK-3 was present, diffusely located throughout the cytoplasm and absent in the nucleus, whereas in meiotic-competent oocytes this cytoplasmic GSK-3 displays a predominant peri-oolemma staining. Treatment of mouse GVI oocytes with lithium chloride (LiCl), which inhibits both inositol monophosphatase (IMPase) and GSK-3, had no significant influence on oocyte viability, morphology, or development to metaphase II (MII). However, LiCl caused abnormal spindle formation and significantly increased incidence of abnormal homologue segregation during the first meiotic division. L690,330, which is a specific IMPase inhibitor, had no significant effect on oocyte viability, morphology, MII development, or homologue segregation. This is the first report of GSK-3 in mammalian oocytes. LiCl inhibition of mouse oocyte GSK-3 modified organization of microtubules and/or function of meiotic spindles thus compromising segregation of condensed bivalent chromosomes.