Enhancing Pharmacy Faculty Well-Being and Productivity While Reducing Burnout.

Objective To explore methods that pharmacy programs can use to redefine their work environment to reduce stress, improve well-being, and increase faculty productivity.Findings To demonstrate a culture of support, organizations should consider a five-fold approach to enhancing and maintaining faculty well-being, including optimizing faculty and staff support, establishing a faculty development and mentoring program, permitting flexibility in work schedules, improving productivity of meetings, and managing communication tools. Individuals can also take measures to improve their well-being, including controlling email, giving attention to faculty citizenship, implementing stress reduction and coping techniques, and maintaining boundaries between work and home.Summary This article discusses approaches that have been shown to reduce burnout and provides strategies organizations and individuals can implement to improve productivity and faculty well-being. While certain areas, such as faculty wellness and productivity, have been well-studied in the pharmacy and health professions literature, significant gaps were identified in other areas, including alternate work arrangements. In some cases, data from the business sector can be extrapolated to pharmacy education; however, inferences from effective corporate strategies may not be transferable to the culture and expectations of academia. While there is significant overlap between institutional and individual strategies, a culture of communication, collaboration, support, and citizenship is foundational. There is no single strategy that will work for everyone, and flexibility is important to develop an individualized approach.

Professional Student Education and Training During the COVID-19 Pandemic.

Introduction: The ongoing COVID-19 pandemic has presented numerous challenges to education at all levels, but has been particularly challenging for professional schools and other educational sectors that require intensive hands-on training. Those institutions have had to deploy and continuously adapt new learning strategies in response to an ever-changing pandemic landscape over the past two years, while at the same time meeting the rigorous proficiency standards for their students. Methods: This communication describes how two professional schools at Oregon State University, the College of Pharmacy and the Carlson College of Veterinary Medicine, pivoted in response to the COVID-19 pandemic to ensure continuity in student training. The adaptations included technological solutions, physical distancing, barriers, reduced group size and scheduling changes in the curriculum, and enhanced personal protective equipment. Results: The available evidence suggest that the biosafety measures implemented to reduce the risk of COVID-19 in the hands-on educational setting appear to have been effective in preventing transmission during classroom and experiential learning activities. Professional licensing exam scores for the students of both colleges remain as high as pre-pandemic values, suggesting that the implemented changes in instruction did not have a detrimental impact on student learning. The scores will need to be monitored for several more years before firm conclusions can be drawn. Discussion: Both colleges implemented creative solutions to the delivery of hands-on pedagogy that sought to balance risk of infection and the necessity to master critical skills that can only be acquired by active learning.

Gene Expression Profiling of Skeletal Muscles.

Next-generation sequencing provides an opportunity for an in-depth biocomputational analysis to identify gene expression patterns between soleus and tibialis anterior, two well-characterized skeletal muscles, and analyze their gene expression profiling. RNA read counts were analyzed for differential gene expression using the R package edgeR. Differentially expressed genes were filtered using a false discovery rate of less than 0.05 c, a fold-change value of more than twenty, and an association with overrepresented pathways based on the Reactome pathway over-representation analysis tool. Most of the differentially expressed genes associated with soleus are coded for components of lipid metabolism and unique contractile elements. Differentially expressed genes associated with tibialis anterior encoded mostly for glucose and glycogen metabolic pathway regulatory enzymes and calcium-sensitive contractile components. These gene expression distinctions partly explain the genetic basis for skeletal muscle specialization, and they may help to explain skeletal muscle susceptibility to disease and drugs and further refine tissue engineering approaches.

Mifepristone and PGF2α activate phosphatidylinositol hydrolysis in the ovine corpus luteum.

Two experiments were conducted to determine whether mifepristone (RU486) and PGF2α activate the phosphatidylinositol hydrolysis pathway during the midluteal phase of the ovine estrous cycle. In experiment 1, ewes on day 8 of the cycle were given 10 µg RU486 or vehicle into the ovarian artery with removal of the corpus luteum (CL) after 10 min. Blood collected prior to and after treatment was analyzed for progesterone. Aliquots of CL were incubated with 10 µCi of 3H-inositol and in the presence and absence of PGF2α (10 nM) for 15 min. Exposure of CL to RU486 and PGF2α increased phosphatidylinositol hydrolysis (p < 0.05). Serum progesterone was reduced in both control and RU486-treated ewes (p < 0.05) compared to concentrations before treatments. In experiment 2, aliquots of CL collected from ewes on day 8 of the cycle were incubated with 3H-inositol and exposed to RU486 (2 µM) in the presence and absence of PGF2α (1 µM) for 15 min. Treatments stimulated phosphatidylinositol hydrolysis as in Exp 1 (p < 0.05). Progesterone concentrations in incubation medium were increased in response to RU486 and PGF2α (p < 0.05). Collectively, these data suggest that RU486 and PGF2α act to stimulate phosphatidylinositol hydrolysis in the mature ovine CL.

A de novo substitution in BCL11B leads to loss of interaction with transcriptional complexes and craniosynostosis.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4-MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4-MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Alteration of Bcl11b upon stimulation of both the MAP kinase- and Gsk3-dependent signaling pathways in double-negative thymocytes.

B-cell lymphoma/leukemia 11B (Bcl11b) is a transcription factor critical for thymocyte development. We have previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double-positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double-negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt-Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and (or) Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation-associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.

FACS-Seq analysis of Pax3-derived cells identifies non-myogenic lineages in the embryonic forelimb.

Skeletal muscle in the forelimb develops during embryonic and fetal development and perinatally. While much is known regarding the molecules involved in forelimb myogenesis, little is known about the specific mechanisms and interactions. Migrating skeletal muscle precursor cells express Pax3 as they migrate into the forelimb from the dermomyotome. To compare gene expression profiles of the same cell population over time, we isolated lineage-traced Pax3+ cells (Pax3 EGFP ) from forelimbs at different embryonic days. We performed whole transcriptome profiling via RNA-Seq of Pax3+ cells to construct gene networks involved in different stages of embryonic and fetal development. With this, we identified genes involved in the skeletal, muscular, vascular, nervous and immune systems. Expression of genes related to the immune, skeletal and vascular systems showed prominent increases over time, suggesting a non-skeletal myogenic context of Pax3-derived cells. Using co-expression analysis, we observed an immune-related gene subnetwork active during fetal myogenesis, further implying that Pax3-derived cells are not a strictly myogenic lineage, and are involved in patterning and three-dimensional formation of the forelimb through multiple systems.

Differential gene regulatory networks in development and disease.

Gene regulatory networks, in which differential expression of regulator genes induce differential expression of their target genes, underlie diverse biological processes such as embryonic development, organ formation and disease pathogenesis. An archetypical systems biology approach to mapping these networks involves the combined application of (1) high-throughput sequencing-based transcriptome profiling (RNA-seq) of biopsies under diverse network perturbations and (2) network inference based on gene-gene expression correlation analysis. The comparative analysis of such correlation networks across cell types or states, differential correlation network analysis, can identify specific molecular signatures and functional modules that underlie the state transition or have context-specific function. Here, we review the basic concepts of network biology and correlation network inference, and the prevailing methods for differential analysis of correlation networks. We discuss applications of gene expression network analysis in the context of embryonic development, cancer, and congenital diseases.

Mapping the Chromatin State Dynamics in Myoblasts.

BACKGROUND: Genome-wide mapping reveals chromatin landscapes unique to cell states. Histone marks of regulatory genes involved in cell specification and organ development provide a powerful tool to map regulatory sequences. H3K4me3 marks promoter regions; H3K27me3 marks repressed regions, and Pol II presence indicates active transcription. The presence of both H3K4me3 and H3K27me3 characterize poised sequences, a common characteristic of genes involved in pattern formation during organogenesis. RESULTS: We used genome-wide profiling for H3K27me3, H3K4me3, and Pol II to map chromatin states in mouse embryonic day 12 forelimbs in wild type (control) and Pitx2-null mutant mice. We compared these data with previous gene expression studies from forelimb Lbx1+ migratory myoblasts and correlated Pitx2-dependent expression profiles and chromatin states. During forelimb development, several lineages including myoblast, osteoblast, neurons, angioblasts etc., require synchronized growth to form a functional limb. We identified 125 genes in the developing forelimb that are Pitx2-dependent. Genes involved in muscle specification and cytoskeleton architecture were positively regulated, while genes involved in axonal path finding were poised. CONCLUSION: Our results have established histone modification profiles as a useful tool for identifying gene regulatory states in muscle development, and identified the role of Pitx2 in extending the time of myoblast progression, promoting formation of sarcomeric structures, and suppressing attachment of neuronal axons.

Kinetic analysis of BCL11B multisite phosphorylation-dephosphorylation and coupled sumoylation in primary thymocytes by multiple reaction monitoring mass spectroscopy.

Transcription factors with multiple post-translational modifications (PTMs) are not uncommon, but comprehensive information on site-specific dynamics and interdependence is comparatively rare. Assessing dynamic changes in the extent of PTMs has the potential to link multiple sites both to each other and to biological effects observable on the same time scale. The transcription factor and tumor suppressor BCL11B is critical to three checkpoints in T-cell development and is a target of a T-cell receptor-mediated MAP kinase signaling. Multiple reaction monitoring (MRM) mass spectroscopy was used to assess changes in relative phosphorylation on 18 of 23 serine and threonine residues and sumoylation on one of two lysine resides in BCL11B. We have resolved the composite phosphorylation-dephosphorylation and sumoylation changes of BCL11B in response to MAP kinase activation into a complex pattern of site-specific PTM changes in primary mouse thymocytes. The site-specific resolution afforded by MRM analyses revealed four kinetic patterns of phosphorylation and one of sumoylation, including both rapid simultaneous site-specific increases and decreases at putative MAP kinase proline-directed phosphorylation sites, following stimulation. These data additionally revealed a novel spatiotemporal bisphosphorylation motif consisting of two kinetically divergent proline-directed phosphorylation sites spaced five residues apart.

Regulation of transcription factor activity by interconnected post-translational modifications.

Transcription factors comprise just over 7% of the human proteome and serve as gatekeepers of cellular function, integrating external signal information into gene expression programs that reconfigure cellular physiology at the most basic levels. Surface-initiated cell signaling pathways converge on transcription factors, decorating these proteins with an array of post-translational modifications (PTMs) that are often interdependent, being linked in time, space, and combinatorial function. These PTMs orchestrate every activity of a transcription factor over its entire lifespan--from subcellular localization to protein-protein interactions, sequence-specific DNA binding, transcriptional regulatory activity, and protein stability--and play key roles in the epigenetic regulation of gene expression. The multitude of PTMs of transcription factors also offers numerous potential points of intervention for development of therapeutic agents to treat a wide spectrum of diseases. We review PTMs most commonly targeting transcription factors, focusing on recent reports of sequential and linked PTMs of individual factors.

CXCR2 macromolecular complex in pancreatic cancer: a potential therapeutic target in tumor growth.

The signaling mediated by the chemokine receptor CXC chemokine receptor 2 (CXCR2) plays an important role in promoting the progression of many cancers, including pancreatic cancer, one of the most lethal human malignancies. CXCR2 possesses a consensus PSD-95/DlgA/ZO-1 (PDZ) motif at its carboxyl termini, which might interact with potential PDZ scaffold/adaptor proteins. We have previously reported that CXCR2 PDZ motif-mediated protein interaction is an important regulator for neutrophil functions. Here, using a series of biochemical assays, we demonstrate that CXCR2 is physically coupled to its downstream effector phospholipase C-ß3 (PLC-ß3) that is mediated by PDZ scaffold protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) into a macromolecular signaling complex both in vitro and in pancreatic cancer cells. We also observe that disrupting the CXCR2 complex, by gene delivery or peptide delivery of exogenous CXCR2 C-tail, significantly inhibits the biologic functions of pancreatic cancer cells (i.e., proliferation and invasion) in a PDZ motif-dependent manner. In addition, using a human pancreatic tumor xenograft model, we show that gene delivery of CXCR2 C-tail sequence (containing the PDZ motif) by adeno-associated virus type 2 viral vector potently suppresses human pancreatic tumor growth in immunodeficient mice. In summary, our results suggest the existence of a physical and functional coupling of CXCR2 and PLC-ß3 mediated through NHERF1, forming a macromolecular complex that is critical for efficient and specific CXCR2 signaling in pancreatic cancer progression. Disrupting this CXCR2 complex could represent a novel and effective treatment strategy against pancreatic cancer.

Grp1-associated scaffold protein (GRASP) is a regulator of the ADP ribosylation factor 6 (Arf6)-dependent membrane trafficking pathway.

GRASP interacts with Grp1 (general receptor for phosphoinositides 1; cytohesin 3), which catalyses nucleotide exchange on and activation of Arf6 (ADP-ribosylation factor-6). Arf6 is a low-molecular-mass GTPase that regulates key aspects of endocytic recycling pathways. Overexpressed GRASP accumulated in the juxtanuclear ERC (endocytic recycling compartment). GRASP co-localized with a constitutively inactive mutant of Arf6 in the ERC such that it was reversed by expression of wild-type Grp1. Co-expression of GRASP and Grp1 promoted membrane ruffling, a cellular hallmark of Arf6 activation. GRASP accumulation in ERC was found to block recycling of the MHC-I (major histocompatibility complex-I), which is trafficked by the Arf6-dependent pathway. In contrast, overexpression of GRASP had no effect on the recycling of transferrin receptors, which are trafficked by a clathrin-dependent pathway. The findings suggest that GRASP regulates the non-clathrin/Arf6-dependent, plasma membrane recycling and signalling pathways.

Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation.

The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa.

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [(3)H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT)>OT≈vasotocin (VT)>vasopressin (VP)>isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT>OT>VT≈VP>IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT>VP>MT≈OT>IT. For both receptors, stimulation of IP accumulation was blocked by d(CH(2))(5)[Tyr(Me)(2)]AVP (Manning compound) and d(CH(2))(5)[Tyr(Me)(2),Thr(4),Tyr-NH(2)]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC(50) of Manning compound was lower for the tMTR (27nM±13) than the tVTR-1a (586nM±166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.

Berberine possesses muscarinic agonist-like properties in cultured rodent cardiomyocytes.

Berberine, a natural product alkaloid, has been shown to display a wide array of pharmacological effects. Generally, the mechanism of action of each of these effects has not been well described. The aim of the present study is to test the hypothesis that some of berberine's cardiovascular effects are mediated through activation of cardiac M2 muscarinic cholinergic receptors. In our studies, we tested the ability of berberine to alter the contraction rate of cultured neonatal rodent cardiomyocytes. In these spontaneously contracting primary cultured cells, berberine reduced the contraction rate in a manner independent of ß-adrenergic receptor blockade but sensitive to pertussis toxin, a Gi/o G protein inhibitor. Muscarinic antagonists completely blocked the effect of berberine on contraction rate of cardiomyocytes, whereas the effect of berberine was not opposed by antagonists to opioid, adenosine or α-adrenergic receptors. Further, berberine bound to muscarinic receptors of adult mouse heart membranes with relatively high affinity (K(i)=5.4×10(-6)M) comparable to that of the classic muscarinic agonist, carbachol, and to muscarinic M2 receptors exogenously expressed in HEK 293 cells (K(i)=4.9×10(-6)M). Therefore, the findings of the present study suggest that berberine is a muscarinic agonist at M2 receptors, potentially explaining some of its reported cardiovascular effects.

Phospholipase Cbeta1b associates with a Shank3 complex at the cardiac sarcolemma.

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell models. Our previous studies have shown that responses to activated Gq in cardiomyocytes are mediated exclusively by phospholipase Cß1b (PLCß1b), because only this PLCß subtype localizes at the cardiac sarcolemma. In the current study, we investigated the proteins involved in targeting PLCß1b to the sarcolemma in neonatal rat cardiomyocytes. PLCß1b, but not PLCß1a, coimmunoprecipitated with the high-MW scaffolding protein SH3 and ankyrin repeat protein 3 (Shank3), as well as the known Shank3-interacting protein α-fodrin. The 32-aa splice-variant-specific C-terminal tail of PLCß1b also associated with Shank3 and α-fodrin, indicating that PLCß1b binds via the C-terminal sequence. Shank3 colocalized with PLCß1b at the sarcolemma, and both proteins were enriched in the light membrane fractions. Knockdown of Shank3 using siRNA reduced PLC activation and downstream hypertrophic responses, demonstrating the importance of sarcolemmal localization for PLC signaling. These data indicate that PLCß1b associates with a Shank3 complex at the cardiac sarcolemma via its splice-variant-specific C-terminal tail. Sarcolemmmal localization is central to PLC activation and subsequent downstream signaling following Gq-coupled receptor activation.

Selective activation of the "b" splice variant of phospholipase Cbeta1 in chronically dilated human and mouse atria.

Atrial fibrillation (AF) is commonly associated with chronic dilatation of the left atrium, both in human disease and animal models. The immediate signaling enzyme phospholipase C (PLC) is activated by mechanical stretch to generate the Ca2+-releasing messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and sn-1,2-diacylglycerol (DAG), an activator of protein kinase C subtypes. There is also evidence that heightened activity of PLC, caused by the receptor coupling protein Gq, can contribute to atrial remodelling. We examined PLC activation in right and left atrial appendage from patients with mitral valve disease (VHD) and in a mouse model of dilated cardiomyopathy caused by transgenic overexpression of the stress-activated protein kinase, mammalian sterile 20 like kinase 1 (Mst1) (Mst1-TG). PLC activation was heightened 6- to 10-fold in atria from VHD patients compared with right atrial tissue from patients undergoing coronary artery bypass surgery (CABG) and was also heightened in the dilated atria from Mst1-TG. PLC activation in human left atrial appendage and in mouse left atria correlated with left atrial size, implying a relationship between PLC activation and chronic dilatation. Dilated atria from human and mouse showed heightened expression of PLCbeta1b, but not of other PLC subtypes. PLCbeta1b, but not PLCbeta1a, caused apoptosis when overexpressed in neonatal rat cardiomyocytes, suggesting that PLCbeta1b may contribute to chamber dilatation. The activation of PLCbeta1b is a possible therapeutic target to limit atrial remodelling in VHD patients.

Gq-initiated cardiomyocyte hypertrophy is mediated by phospholipase Cbeta1b.

Activation of the heterotrimeric G protein Gq causes cardiomyocyte hypertrophy in vivo and in cell culture models. Hypertrophic responses induced by pressure or volume overload are exacerbated by increased Gq activity and ameliorated by Gq inhibition. Gq activates phospholipase Cbeta (PLCbeta) subtypes, resulting in generation of the intracellular messengers inositol(1,4,5)tris-phosphate [Ins(1,4,5)P(3)] and sn-1,2-diacylglycerol (DAG), which regulate intracellular Ca(2+) and conventional protein kinase C subtypes, respectively. Gq can also signal independently of PLCbeta, and the involvement of either Ins(1,4,5)P(3) or DAG in cardiomyocyte hypertrophy has not been unequivocally established. Overexpression of one splice variant of PLCbeta1, specifically PLCbeta1b, in neonatal rat cardiomyocytes causes increased cell size, elevated protein/DNA ratio, and heightened expression of the hypertrophy-related marker gene, atrial natriuretic peptide. The other splice variant, PLCbeta1a, had no effect. Expression of a 32-aa C-terminal PLCbeta1b peptide, which competes with PLCbeta1b for sarcolemmal association, prevented PLC activation and eliminated hypertrophic responses initiated by Gq or Gq-coupled alpha(1)-adrenergic receptors. In contrast, a PLCbeta1a C-terminal peptide altered neither PLC activity nor cellular hypertrophy. We conclude that hypertrophic responses initiated by Gq are mediated specifically by PLCbeta1b. Preventing PLCbeta1b association with the sarcolemma may provide a useful therapeutic target to limit hypertrophy.

PI(3,4,5)P3 potentiates phospholipase C-beta activity.

Phospholipase C-beta (PLC-beta) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-beta1 and PLC-beta3 bound immobilized PIP(3). In this study, PIP(3) was found to potentiate Ca(2+)-stimulated PLC-beta activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP(3) accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP(3) accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP(3) in directly potentiating PLC-beta activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-beta3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-beta is not important for p110CAAX-induced membrane association.