Raj Wadgaonkar

Murine Double Minute (MDM2) Blocks p53-coactivator Interaction, a New Mechanism for Inhibition of p53-dependent Gene Expression

The ability of the p53 tumor suppressor to induce cell cycle arrest and cell death is closely regulated under normal conditions. The transcriptional activity of p53 is negatively controlled by murine double minute (MDM2). p53 requires the coactivator CREB-binding protein (CBP), or its structural homolog, p300, to stimulate transcription of responsive genes. Here we find that the transactivation domain of p53 selectively interacts with the N- and C-terminal regions of CBP/p300. A mutant CBP lacking the N terminus failed to stimulate p53-dependent transactivation. In both p53 null Saos2 cells, and in UV-irradiated MCF7 cells, we observed that MDM2 associates with the N-terminal region of CBP/p300. Because p53 interacts with both MDM2 and CBP/p300 through its trans-activation domain, we examined the role of MDM2 in p53-coactivator interactions. MDM2 blocked CBP/p300 recruitment in vitro and inhibited the interaction of the transactivating region of p53 with both the N- or C-terminal regions of CBP/p300 in a mammalian two-hybrid assay. These observations suggest that MDM2 may be inhibiting p53 trans-activation by shielding its activation domain from the coactivators, a new mechanism for the inhibition of p53-dependent gene expression.

Caspase-dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF-α-mediated bovine pulmonary endothelial cell apoptosis

Cytoskeletal proteins are key participants in the cellular progression to apoptosis. Our previous work demonstrated the critical dependence of actomyosin rearrangement and MLC phosphorylation in TNF‐α‐induced endothelial cell apoptosis. As these events reflect the activation of the multifunctional endothelial cell (EC) MLCK isoform, we assessed the direct role of EC MLCK in the regulation of TNF‐α‐induced apoptosis. Bovine pulmonary artery endothelial cells expressing either an adenovirus encoding antisense MLCK cDNA (Ad.GFP‐AS MLCK) or a dominant/negative EC MLCK construct (EC MLCK‐ATP‐del) resulted in marked reductions in MLCK activity and TNF‐α‐mediated apoptosis. In contrast, a constitutively active EC MLCK lacking the carboxyl‐terminal autoinhibitory domains (EC MLCK‐1745) markedly enhanced the apoptotic response to TNF‐α. Immunostain‐ing in GFP‐EC MLCK‐expressing cells revealed colocalization of caspase 8 and EC MLCK along actin stress fibers after TNF‐α. TNF‐α induced the caspase‐dependent cleavage of EC MLCK‐1745 in transfected endothelial cells, which was confirmed by mass spectroscopy with in vitro cleavage by caspase 3 at LKKD (D1703). The resulting MLCK fragments displayed significant calmodulin‐independent kinase activity. These studies convincingly demonstrate that novel interactions between the apoptotic machinery and EC MLCK exist that regulate the endothelial contractile apparatus in TNF‐α‐induced apoptosis.—Petrache, I., Birukov, K., Zaiman, A. L., Crow, M. T., Deng, H., Wadgaonkar, R., Romer, L. H., Garcia, J. G. N. Caspase‐dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF‐α‐mediated bovine pulmonary endothelial cell apoptosis. FASEB J. 17, 407–416 (2003)

Differential regulation of sphingosine kinases 1 and 2 in lung injury.

Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Endothelial cell myosin light chain kinase (MLCK) regulates TNFα‐induced NFκB activity

Tumor necrosis factor (TNFα‐) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa‐B (NFκB) activation, a regulator of anti‐apoptotic genes. We previously demonstrated that increased EC contractility, rearrangement of the actin cytoskeleton, and increased myosin light chain (MLC) phosphorylation occurs as a consequence of TNFα‐induced activation of EC MLC kinase (EC MLCK) and is required for bovine lung EC apoptosis. As the association between MLCK and pro‐survival signals such as NFκB activation is unknown, we studied the role of MLCK in the regulation of NFκB‐dependent transactivation in bovine pulmonary artery EC. Both TNFα‐induced increase in NFκB dependent transactivation measured by NFκB luciferase reporter assay (∼ fivefold) and nuclear translocation of NFκB were significantly inhibited by MLCK‐selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease). Furthermore, our data revealed that inhibition of MLCK attenuated the TNFα‐induced IκB phosphorylation, translocation of p65, NFκB‐DNA binding, and NFκB transcriptional activity. Molecular approaches to either reduce EC MLCK expression (AdV EC MLCK antisense construct) or to reduce kinase activity (kinase‐dead EC MLCK ATPdel mutant) produced similar attenuation of the TNFα‐induced NFκB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFα‐induced luciferase activity. Together, these novel observations indicate that TNFα‐induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFα‐dependent NFκB activation and amplification of pro‐survival signals.

Congenital heart block: Identification of autoantibody binding site on the extracellular loop (domain I, S5–S6) of α1D L-type Ca channel

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit alpha(1D) L-type Ca current, I(Ca-L), and cross-react with the alpha(1D) Ca channel protein. This study aims at identifying the possible binding sites on alpha(1D) protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four alpha(1D) Ca channel protein domains I-IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5-S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (-anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the alpha(1D) I(Ca-L) expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel alpha(1D) subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB.

A transgenic mouse with vascular endothelial over-expression of the non-muscle myosin light chain kinase-2 isoform is susceptible to inflammatory lung injury: role of sexual dimorphism and age

We have generated genetically engineered mice that are uniquely susceptible to lipopolysaccharide (LPS)-induced and mechanical ventilation-induced lung injury in a sex-specific and age-specific manner. These mice express a nonmuscle isoform of the myosin light chain kinase gene (nmMLCK2) targeted to the endothelium. Homozygous mice have significantly reduced fecundity and litter survival until weaning, and they are initially growth delayed but eventually exceed the size of wild-type littermates. Mice at all ages show increased protein transport across the lung barrier; however, the phenotype is most discernible in 8-12-week-old male mice. When subjected to a clinically relevant LPS-induced lung injury model, 8-12-week-old young females and 30-36-week-old males seem to be the most significantly injured group. In contrast, 30-36-week-old males remain the most significantly injured group when mechanically ventilated at high tidal volumes, which is a clinically relevant model of mechanical stress lung injury. These data reveal that nmMLCK2 overexpression in the endothelium exacerbates lung injury in vivo in a sexually dimorphic and age-dependent manner.

Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium.

The endothelial cell Ca2+/calmodulin (CaM)‐dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N‐terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two‐hybrid assay system using the entire EC MLCK1 N‐terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non‐denaturing conditions and by GST pull down experiments using GST‐N‐terminal MLCK (#1–923) and MLCK N‐terminal deletion mutants in TNFα‐ and thrombin‐stimulated endothelium. This EC MLCK–MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFα‐ and thrombin‐stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope‐tagged, full‐length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non‐muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.

Sphingosine Kinase 1 Deficiency Exacerbates LPS-Induced Neuroinflammation

The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1−/− mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1−/− mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury. Research Highlights • Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. • Sphingosine kinase 1 deletion worsens the effect of the LPS. • Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.

Pancreatitis-Associated Protein 2 Modulates Inflammatory Responses in Macrophages

Pancreatitis-associated proteins (PAP) are stress-induced secretory proteins that are implicated in immunoregulation. Previous studies have demonstrated that PAP is up-regulated in acute pancreatitis and that gene knockdown of PAP correlated with worsening severity of pancreatitis, suggesting a protective effect for PAP. In the present study, we investigated the effect of PAP2 in the regulation of macrophage physiology. rPAP2 administration to clonal (NR8383) and primary macrophages were followed by an assessment of cell morphology, inflammatory cytokine expression, and studies of cell-signaling pathways. NR8383 macrophages which were cultured in the presence of PAP2 aggregated and exhibited increased expression of IL-1, IL-6, TNF-α, and IL-10; no significant change was observed in IL-12, IL-15, and IL-18 when compared with controls. Chemical inhibition of the NFκB pathway abolished cytokine production and PAP facilitated nuclear translocation of NF-κB and phosphorylation of IκBα inhibitory protein suggesting that PAP2 signaling involves this pathway. Cytokine responses were dose dependent. Interestingly, similar findings were observed with primary macrophages derived from lung, peritoneum, and blood but not spleen. Furthermore, PAP2 activity was inhibited by the presence of serum, inhibition which was overcome with increased PAP2. Our results demonstrate a new function for PAP2: it stimulates macrophage activity and likely modulates the inflammatory environment of pancreatitis.

Mutation analysis of the non‐muscle myosin light chain kinase (MLCK) deletion constructs on CV1 fibroblast contractile activity and proliferation

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N‐terminal actin binding domain, a central kinase domain, and C‐terminal calmodulin‐ and myosin‐binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel‐amino‐terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non‐muscle cells, we constructed two epitope‐tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C‐terminal auto‐inhibitory and calmodulin‐binding domain (EC MLCK1745) or the ATP‐binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF‐α) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co‐localized with myosin light chains (MLCs) diphosphorylated on Ser19 and Thr18. In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin‐ and TNFα‐induced stress fiber formation and MLC phosphorylation, suggesting this kinase‐dead mutant functions as a dominant‐negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum‐stimulated growth rate of mutant MLCK‐transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.