Jianfen Guo

Protease-Activated Receptor-1–Mediated DNA Synthesis in Cardiac Fibroblast Is via Epidermal Growth Factor Receptor Transactivation: Distinct PAR-1 Signaling Pathways in Cardiac Fibroblasts and Cardiomyocytes

Abstract— Proteases elaborated by inflammatory cells in the heart would be expected to drive cardiac fibroblasts to proliferate, but protease-activated receptor (PAR) function in cardiac fibroblasts has never been considered. This study demonstrates that PAR-1 is the only known PAR family member functionally expressed by cardiac fibroblasts and that PAR-1 activation by thrombin leads to increased DNA synthesis in cardiac fibroblasts. The increase in DNA synthesis induced by PAR-1 substantially exceeds the effects of other G protein–coupled receptor agonists in this cell type. PAR-1 stimulates phosphoinositide hydrolysis and mobilizes intracellular calcium via pertussis toxin (PTX)-sensitive and PTX-insensitive pathways. Activation of PAR-1 leads to an increase in Src, Fyn, and epidermal growth factor receptor (EGFR) phosphorylation, with EGFR receptor transactivation by Src family kinases the major mechanism for PAR-1–dependent activation of extracellular signal–regulated kinase, p38-mitogen-activated protein kinase, and protein kinase B. Activation of PAR-1 also leads to an increase in DNA synthesis. PAR-1 signaling is highly contextual in nature, inasmuch as PAR-1 activates extracellular signal–regulated kinase and only weakly stimulates protein kinase B via a pathway that does not involve EGFR transactivation in cardiomyocytes. PAR-1 responses in cardiac fibroblasts and cardiomyocytes are predicted to contribute importantly to remodeling during cardiac injury and/or inflammation.

p66Shc Links α1-Adrenergic Receptors to a Reactive Oxygen Species–Dependent AKT-FOXO3A Phosphorylation Pathway in Cardiomyocytes

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an &agr;1-adrenergtic receptor (&agr;1-AR) pathway that requires the cooperative effects of protein kinase (PK)Cϵ and PKC&dgr; and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. &agr;1-ARs promote p66Shc-YY239/240 phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCϵ activity. &agr;1-ARs also increase p66Shc-S36 phosphorylation via an EGFR transactivation pathway involving PKC&dgr;. p66Shc links &agr;1-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the &agr;1-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference–mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel &agr;1-AR– and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser744/Ser748 and Ser916

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.

Protein kinase Cepsilon (PKCepsilon) and Src control PKCdelta activation loop phosphorylation in cardiomyocytes.

Protein kinase Cδ (PKCδ) is unusual among AGC kinases in that it does not require activation loop (Thr505) phosphorylation for catalytic competence. Nevertheless, Thr505 phosphorylation has been implicated as a mechanism that influences PKCδ activity. This study examines the controls of PKCδ-Thr505 phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCδ autophosphorylation in the “priming” maturational PKCδ-Thr505 phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCδ-Thr505 phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the α1-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCϵ overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCδ-Thr505 autophosphorylation and PKCδ-Thr505 phosphorylation in trans by PKCϵ. Our studies show that adenovirus-mediated PKCϵ overexpression leads to an increase in PKCδ-Thr505 phosphorylation. However, this cannot be attributed to an effect of PKCϵ to function as a direct PKCδ-Thr505 kinase, since the PKCϵ-dependent increase in PKCδ-Thr505 phosphorylation is accompanied by (and dependent upon) increased PKCδ phosphorylation at Tyr311 and Tyr332. Further studies implicate Src in this mechanism, showing that 1) PKCϵ overexpression increases PKCδ-Thr505 phosphorylation in cardiomyocytes and Src+ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCδ-Tyr311/Tyr332 phosphorylation), and 2) in vitro PKCδ-Thr505 autophosphorylation is augmented in assays performed with Src (which promotes PKCδ-Tyr311/Tyr332 phosphorylation). Collectively, these results identify a novel PKCδ-Thr505 autophosphorylation mechanism that is triggered by PKCϵ overexpression and involves Src-dependent PKCδ-Tyr311/Tyr332 phosphorylation.

Protein Kinase D Links Gq-coupled Receptors to cAMP Response Element-binding Protein (CREB)-Ser133 Phosphorylation in the Heart

Many growth regulatory stimuli promote cAMP response element-binding protein (CREB) Ser133 phosphorylation, but the physiologically relevant CREB-Ser133 kinase(s) in the heart remains uncertain. This study identifies a novel role for protein kinase D (PKD) as an in vivo cardiac CREB-Ser133 kinase. We show that thrombin activates a PKCδ-PKD pathway leading to CREB-Ser133 phosphorylation in cardiomyocytes and cardiac fibroblasts. α1-Adrenergic receptors also activate a PKCδ-PKD-CREB-Ser133 phosphorylation pathway in cardiomyocytes. Of note, while the epidermal growth factor (EGF) promotes CREB-Ser133 phosphorylation via an ERK-RSK pathway in cardiac fibroblasts, the thrombin-dependent EGFR transactivation pathway leading to ERK-RSK activation does not lead to CREB-Ser133 phosphorylation in this cell type. Adenoviral-mediated overexpression of PKCδ (but not PKCϵ or PKCα) activates PKD; PKCδ and PKD1-S744E/S748E overexpression both promote CREB-Ser133 phosphorylation. Pasteuralla multocida toxin (PMT), a direct Gαq agonist that induces robust cardiomyocyte hypertrophy, also activates the PKD-CREB-Ser133 phosphorylation pathway, leading to the accumulation of active PKD and Ser133-phosphorylated CREB in the nucleus, activation of a CRE-responsive promoter, and increased Bcl-2 (CREB target gene) expression in cardiomyocyte cultures. Cardiac-specific Gαq overexpression also leads to an increase in PKD-Ser744/Ser748 and CREB-Ser133 phosphorylation as well as increased Bcl-2 protein expression in the hearts of transgenic mice. Collectively, these studies identify a novel Gαq-PKCδ-PKD-CREB-Ser133 phosphorylation pathway that is predicted to contribute to cardiac remodeling and could be targeted for therapeutic advantage in the setting of heart failure phenotypes.

Reactive Oxygen Species Decrease cAMP Response Element Binding Protein Expression in Cardiomyocytes via a Protein Kinase D1-Dependent Mechanism That Does Not Require Ser133 Phosphorylation

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser133 in cardiomyocytes. Although CREB-Ser133 phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser133 phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser133 phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

c-Cbl Ubiquitin Ligase Regulates Focal Adhesion Protein Turnover and Myofibril Degeneration Induced by Neutrophil Protease Cathepsin G

Background: The neutrophil protease cathepsin G induces myocyte anoikis. Results: Cathepsin G promotes c-Cbl activation and interaction with focal adhesion proteins that leads to focal adhesion and myofibril protein degradation and myocyte anoikis. Conclusion: c-Cbl is a key ligase required during cathepsin G-induced focal adhesion and myofibrillar protein degradation. Significance: This is a novel mechanism to regulate focal adhesion and myofibril stability and turnover. The neutrophil-derived serine protease, cathepsin G (Cat.G), has been shown to induce myocyte detachment and apoptosis by anoikis through down-regulation of focal adhesion (FA) signaling. However, the mechanisms that control FA protein stability and turnover in myocytes are not well understood. Here, we have shown that the Casitas b-lineage lymphoma (c-Cbl), adaptor protein with an intrinsic E3 ubiquitin ligase activity, is involved in FA and myofibrillar protein stability and turnover in myocytes. Cat.G treatment induced c-Cbl activation and its interaction with FA proteins. Deletion of c-Cbl using c-Cbl knock-out derived myocytes or inhibition of c-Cbl ligase activity significantly reduced FA protein degradation, myofibrillar degeneration, and myocyte apoptosis induced by Cat.G. We also found that inhibition of the proteasome activity, but not the lysosome or the calpain activity, markedly attenuated FA and myofibrillar protein degradation induced by Cat.G. Interestingly, c-Cbl activation induced by Cat.G was mediated through epidermal growth factor receptor (EGFR) transactivation as inhibition of EGFR kinase activity markedly attenuated c-Cbl phosphorylation and FA protein degradation induced by Cat.G. These findings support a model in which neutrophil protease Cat.G promotes c-Cbl interaction with FA proteins, resulting in enhanced c-Cbl-mediated FA protein ubiquitination and degradation, myofibril degradation, and subsequent down-regulation of myocyte survival signaling.

α1-Adrenergic Receptors Activate AKT via a Pyk2/PDK-1 Pathway That Is Tonically Inhibited by Novel Protein Kinase C Isoforms in Cardiomyocytes

AKT is a potent antiapoptotic kinase, but its role in the cardioprotective actions of α1-adrenergic receptors (ARs) remains uncertain, because α1-ARs typically induce little-to-no AKT activation in most cardiomyocyte models. This study identifies a prominent α1-AR–dependent AKT activation pathway that is under tonic inhibitory control by novel protein kinase Cs (nPKCs) in neonatal rat cardiomyocyte cultures. We also implicate Pyk2, Pyk2 complex formation with PDK-1 and paxillin, and increased PDK-1–Y373/376 phosphorylation as the mechanism that links α1-AR activation to increased AKT phosphorylation. nPKCs (which are prominent α1-AR effectors) interfere with this α1-AR–dependent AKT activation by blocking Pyk2/PDK-1/paxillin complex formation and PDK-1–Y373/376 phosphorylation. Additional studies used an adenoviral-mediated overexpression strategy to show that Pyk2 exerts dual controls on antiapoptotic PDK-1/AKT and proapoptotic c-Jun N-terminal kinase (JNK) pathways. Although the high nPKC activity of most cardiomyocyte models favors Pyk2 signaling to JNK (and cardiac apoptosis), the cardioprotective actions of Pyk2 through the PDK-1/AKT pathway are exposed when PKC or JNK activation is prevented. Collectively, these studies identify JNK and AKT as functionally distinct downstream components of the α1-AR/Pyk2 signaling pathway. We also implicate nPKCs as molecular switches that control the balance of signaling via proapoptotic JNK and antiapoptotic PDK-1/AKT pathways, exposing a novel mechanism for nPKC-dependent regulation of cardiac hypertrophy and failure.

Protein Kinase D Isoforms Are Activated in an Agonist-specific Manner in Cardiomyocytes

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α1-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α1-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser133 and cTnI-Ser23/Ser24 phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser133 phosphorylation; there is no associated increase in cTnI-Ser23/Ser24 phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser133 phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-Tyr311 Phosphorylation in Cardiomyocyte Caveolae

Protein kinase Cδ (PKCδ) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCδ also is dynamically regulated through tyrosine phosphorylation in H2O2- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H2O2 activates Src and related Src-family kinases (SFKs), which function as dual PKCδ-Tyr311 and -Tyr332 kinases in vitro and contribute to H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCδ-Tyr311 phosphorylation, but this is not associated with SFK activation or PKCδ-Tyr332 phosphorylation. Rather, PMA increases PKCδ-Tyr311 phosphorylation by delivering PKCδ to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCδ-Tyr311 phosphorylation, without blocking H2O2-dependent PKCδ-Tyr311 phosphorylation. The enzyme that acts as a PKCδ-Tyr311 kinase without increasing PKCδ phosphorylation at Tyr332 in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCδ-Tyr311 kinase, PMA-dependent PKCδ-Tyr311 phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCδ to caveolae and increase PKCδ-Tyr311 phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCδ exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCδ localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.