Mary Cherian-Shaw

Granulosa cell expression of G1/S phase cyclins and cyclin-dependent kinases in PMSG-induced follicle growth.

Follicular development involves a complex orchestration of granulosa cell proliferation and differentiation. It is becoming increasingly apparent that the rate of granulosa cell proliferation declines as follicles reach the large antral status, prior to an ovulatory gonadotropin stimulus, although a precise time course and mechanism for this decline has not been described. The goal of the present study was to characterize granulosa cell proliferation following the onset of antral follicle growth in PMSG-primed immature rats, with emphasis on G1/S phase cyclins and cyclin-dependent kinases. Flow cytometric analysis demonstrated that the percentage of granulosa cells in S phase peaked 24-30 h post-PMSG and declined to control levels 48 h after PMSG administration. Expression of both Cyclin D2 and Cdk 4 was highest 12h post-PMSG and decreased to control levels by 48 h. In addition, Cdk 2 protein increased transiently 12-24h after PMSG. Cyclin E expression increased significantly by 12h but remained elevated through 48 h, and multiple isoforms of Cyclin E were observed with increased proliferation. Both Cdk 4 and Cdk 2 activity parallel protein expression, although, changes in Cdk 2 were more marked. Levels of mRNA for the cell cycle inhibitors p21CIP1 and p27KIP1 increased significantly by 48 h post-PMSG. These results demonstrate that PMSG-stimulated movement of granulosa cells across the G1/S boundary during follicle growth is transient. In addition, the control of granulosa cell proliferation may reside through the regulation of both Cdk 2 and Cdk 4.

Expression of Scavenger Receptor-BI and Low-Density Lipoprotein Receptor and Differential Use of Lipoproteins to Support Early Steroidogenesis in Luteinizing Macaque Granulosa Cells

An ovulatory hCG stimulus to rhesus macaques undergoing controlled ovarian stimulation protocols results in a rapid and sustained increase in progesterone synthesis. The use of lipoproteins as a substrate for progesterone synthesis remains unclear, and the expression of lipoprotein receptors [very-low-density lipoprotein receptor (VLDLR), low-density lipoprotein receptor (LDLR), and scavenger receptor-BI (SR-BI)] soon after human chorionic gonadotropin (hCG) (<12 h) has not been characterized. This study investigated lipoprotein receptor expression and lipoprotein (VLDL, LDL, and HDL) support of steroidogenesis during luteinization of macaque granulosa cells. Granulosa cells were aspirated from rhesus monkeys undergoing controlled ovarian stimulation before or up to 24 h after an ovulatory hCG stimulus. The expression of VLDLR decreased within 3 h of hCG, whereas LDLR and SR-BI increased at 3 and 12 h, respectively. Granulosa cells isolated before hCG were cultured for 24 h in the presence of FSH or FSH plus hCG with or without VLDL, LDL, or HDL. Progesterone levels increased in the presence of hCG regardless of lipoprotein addition, although LDL, but not HDL, further augmented hCG-induced progesterone. Other cells were cultured with FSH or FSH plus hCG without an exogenous source of lipoprotein for 24 h, followed by an additional 24 h culture with or without lipoproteins. Cells treated with hCG in the absence of any lipoprotein were unable to maintain progesterone levels through 48 h, whereas LDL (but not HDL) sustained progesterone synthesis. These data suggest that an ovulatory stimulus rapidly mobilizes stored cholesterol esters for use as a progesterone substrate and that as these are depleted, new cholesterol esters are obtained through an LDLR- and/or SR-BI-mediated mechanism.

Intrapolypeptide disulfides in human alphaA-crystallin and their effect on chaperone-like function.

We report studies on the role of protein-protein disulfides (PSSP) in the age-related loss of chaperone activity of α-crystallins. αL-Crystallin fraction was isolated from human lenses of different ages and the chaperone-like activity was determined before and after treatment with glutathione reductase (GR) and NADPH. The results confirmed an age-dependent decrease in chaperone-like function and significant improvement of this function by GR treatment. Electrospray ionization mass spectrometric (ESIMS) analysis of αA-crystallin suggested the presence of very little protein-glutathione mixed disulfides. ESIMS analysis of Asp-N digests of αA-crystallin revealed that nearly all the remaining portion of Cys-131 and Cys-142 of αA-crystallin was present in the form of intrapolypeptide disulfide bonds. These results show for the first time that predominantly disulfide bonds formed during aging contribute to the age-dependent loss in chaperone activity of α-crystallin in human lenses.

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.

Histone deacetylase inhibitors prevent pulmonary endothelial hyperpermeability and acute lung injury by regulating heat shock protein 90 function

Transendothelial hyperpermeability caused by numerous agonists is dependent on heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores transendothelial permeability. Hyperacetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating lipopolysaccharide (LPS)-induced transendothelial hyperpermeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced transendothelial hyperpermeability, acetylated and suppressed Hsp90 chaperone function, and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor RGFP-966 or the HDAC6-selective inhibitor tubastatin A provided partial protection against LPS-mediated transendothelial hyperpermeability. Similarly, knock down of HDAC3 and HDAC6 by specific small-interfering RNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacological inhibition of both HDAC3 and -6 attenuated the inflammation, capillary permeability, and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased transendothelial hyperpermeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.

PKCδ stimulates macropinocytosis via activation of SSH1-cofilin pathway

Macropinocytosis is an actin-dependent endocytic mechanism mediating internalization of extracellular fluid and associated solutes into cells. The present study was designed to identify the specific protein kinase C (PKC) isoform(s) and downstream effectors regulating actin dynamics during macropinocytosis. We utilized various cellular and molecular biology techniques, pharmacological inhibitors and genetically modified mice to study the signaling mechanisms mediating macropinocytosis in macrophages. The qRT-PCR experiments identified PKCδ as the predominant PKC isoform in macrophages. Scanning electron microscopy and flow cytometry analysis of FITC-dextran internalization demonstrated the functional role of PKCδ in phorbol ester- and hepatocyte growth factor (HGF)-induced macropinocytosis. Western blot analysis demonstrated that phorbol ester and HGF stimulate activation of slingshot phosphatase homolog 1 (SSH1) and induce cofilin Ser-3 dephosphorylation via PKCδ in macrophages. Silencing of SSH1 inhibited cofilin dephosphorylation and macropinocytosis stimulation. Interestingly, we also found that incubation of macrophages with BMS-5, a potent inhibitor of LIM kinase, does not stimulate macropinocytosis. In conclusion, the findings of the present study demonstrate a previously unidentified mechanism by which PKCδ via activation of SSH1 and cofilin dephosphorylation stimulates membrane ruffle formation and macropinocytosis. The results of the present study may contribute to a better understanding of the regulatory mechanisms during macrophage macropinocytosis.

Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role

We have previously shown that Gs‐coupled adenosine receptors (A2a) are primarily involved in adenosine‐induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine‐induced Rac1 activation and EC barrier enhancement is mediated by Gs‐dependent stimulation of cAMP‐dependent Epac1‐mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively‐active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine‐induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine‐induced increase in TER. We next examined the role of cAMP‐activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine‐induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine‐induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine‐induced increases in TER and Tiam1 depletion had no effect on adenosine‐induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine‐induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP‐dependent Epac1/Vav2/Rap1‐mediated signaling.

Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration‐approved drugs

Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non‐specific endocytosis‐independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA‐approved drug library.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5′‐[γ‐thio]‐triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS‐induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP‐independent pathway of PKA activation. Furthermore, ATPγS‐induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA‐anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase‐targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS‐induced increases in transendothelial electrical resistance. Ado‐induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP‐dependent manner. In summary, ATPγS‐induced enhancement of the EC barrier is EPAC1‐independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP‐independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Hyper-activation of pp60 Src limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury

Abstract The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite‐mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60Src activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3‐nitrotyrosine (3‐NT) levels. These effects could be simulated by the over‐expression of a constitutively active pp60Src (Y527FSrc) mutant and attenuated by over‐expression of dominant negative pp60Src mutant or reducing pp60Src expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60Src expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over‐expressing PAEC. The effects in PAEC were also recapitulated when we transiently over‐expressed Y527FSrc in the mouse lung. Finally, we found that the pp60‐Src‐mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60Src‐mediated inhibition. We conclude that pp60Src can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI. Graphical abstract Figure. No Caption available. HighlightsThe disruption of the endothelial barrier by LPS involves the uncoupling of eNOS.LPS increases pp60Src activity, NO production, NOS uncoupling, and protein nitration.eNOS uncoupling correlated with a decrease in DDAH activity and an increase in ADMA.The decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207.