Shahab Uddin

Activation of the p38 Mitogen-activated Protein Kinase by Type I Interferons

The p38 mitogen-activated protein (Map) kinase plays a critical role in the generation of signals in response to stress stimuli, but its role in interferon (IFN) signaling and its potential regulatory role in the activation of Jak-signal transducer and activator of transcription (Stat) pathway are not known. In the present study, we provide evidence that the p38 Map kinase is rapidly phosphorylated and activated during treatment of cells with Type I interferons (IFNα and IFNβ). Furthermore, the Type I IFN-dependent activation of p38 regulates induction of the catalytic domains of MapKap kinase-2 and MapKap kinase-3, strongly suggesting the existence of an IFNα signaling cascade activated downstream of the p38 kinase. The engagement of this pathway in interferon signaling plays a critical role in interferon-dependent transcriptional regulation, as evidenced by the fact that inhibition of p38 activation results in abrogation of interferon-dependent gene transcription via interferon-stimulated response elements. Interestingly, inhibition of the kinase activity of the p38 blocks IFNα-induced gene transcription without inhibiting DNA binding or tyrosine phosphorylation of Stat proteins, suggesting that the p38 pathway acts in cooperation with the Stat pathway. Thus, the p38 kinase signaling cascade is activated by the Type I interferon receptor and plays a critical role in interferon signaling and interferon-dependent transcriptional regulation.

Protein kinase c-δ (PKC-δ) is activated by Type I interferons and mediates phosphorylation of Stat1 on serine 727

It is well established that engagement of the Type I interferon (IFN) receptor results in activation of JAKs (Janus kinases), which in turn regulate tyrosine phosphorylation of STAT proteins. Subsequently, the IFN-dependent tyrosine-phosphorylated/activated STATs translocate to the nucleus to regulate gene transcription. In addition to tyrosine phosphorylation, phosphorylation of Stat1 on serine 727 is essential for induction of its transcriptional activity, but the IFNα-dependent serine kinase that regulates such phosphorylation remains unknown. In the present study we provide evidence that PKC-δ, a member of the protein kinase C family of proteins, is activated during engagement of the Type I IFN receptor and associates with Stat1. Such an activation of PKC-δ appears to be critical for phosphorylation of Stat1 on serine 727, as inhibition of PKC-δ activation diminishes the IFNα- or IFNβ-dependent serine phosphorylation of Stat1. In addition, treatment of cells with the PKC-δ inhibitor rottlerin or the expression of a dominant-negative PKC-δ mutant results in inhibition of IFNα- and IFNβ-dependent gene transcription via ISRE or GAS elements. Interestingly, PKC-δ inhibition also blocks activation of the p38 MAP kinase, the function of which is required for IFNα-dependent transcriptional regulation, suggesting a dual mechanism by which this kinase participates in the generation of IFNα responses. Altogether, these findings indicate that PKC-δ functions as a serine kinase for Stat1 and an upstream regulator of the p38 MAP kinase and plays an important role in the induction of Type I IFN-biological responses.

Protein Kinase C-δ Regulates Thrombin-Induced ICAM-1 Gene Expression in Endothelial Cells via Activation of p38 Mitogen-Activated Protein Kinase

ABSTRACT The procoagulant thrombin promotes the adhesion of polymorphonuclear leukocytes to endothelial cells by a mechanism involving expression of intercellular adhesion molecule 1 (ICAM-1) via an NF-κB-dependent pathway. We now provide evidence that protein kinase C-δ (PKC-δ) and the p38 mitogen-activated protein (MAP) kinase pathway play a critical role in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. We observed the phosphorylation of PKC-δ and p38 MAP kinase within 1 min after thrombin challenge of human umbilical vein endothelial cells. Pretreatment of these cells with the PKC-δ inhibitor rottlerin prevented the thrombin-induced phosphorylation of p38 MAP kinase, suggesting that p38 MAP kinase signals downstream of PKC-δ. Inhibition of PKC-δ or p38 MAP kinase by pharmacological and genetic approaches markedly decreased the thrombin-induced NF-κB activity and resultant ICAM-1 expression. The effects of PKC-δ inhibition were secondary to inhibition of IKKβ activation and of subsequent NF-κB binding to the ICAM-1 promoter. The effects of p38 MAP kinase inhibition occurred downstream of IκBα degradation without affecting the DNA binding function of nuclear NF-κB. Thus, PKC-δ signals thrombin-induced ICAM-1 gene transcription by a dual mechanism involving activation of IKKβ, which mediates NF-κB binding to the ICAM-1 promoter, and p38 MAP kinase, which enhances transactivation potential of the bound NF-κB p65 (RelA).

Protein Kinase B/Akt Mediates Effects of Insulin on Hepatic Insulin-like Growth Factor-binding Protein-1 Gene Expression through a Conserved Insulin Response Sequence

Insulin regulates the expression of multiple hepatic genes through a conserved insulin response sequence (IRS) (CAAAAC/TAA) by an as yet undetermined mechanism. Protein kinase B/Akt (PKB/Akt), a member of the PKA/PKC serine/threonine kinase family, functions downstream from phosphatidylinositol 3′-kinase (PI3K) in mediating effects of insulin on glucose transport and glycogen synthesis. We asked whether PKB/Akt mediates sequence-specific effects of insulin on hepatic gene expression using the model of the insulin-like growth factor binding protein-1 (IGFBP-1) promoter. Insulin lowers IGFBP-1 mRNA levels, inhibits IGFBP-1 promoter activity, and activates PKB/Akt in HepG2 hepatoma cells through a PI3K-dependent, rapamycin-insensitive mechanism. Constitutively active PI3K and PKB/Akt are each sufficient to mediate effects of insulin on the IGFBP-1 promoter in a nonadditive fashion. Dominant negative K179 PKB/Akt disrupts the ability of insulin and PI3K to activate PKB/Akt and to inhibit promoter activity. The IGFBP-1 promoter contains two IRSs each of which is sufficient to mediate sequence-specific effects of insulin, PI3K, and PKB/Akt on promoter activity. Highly related IRSs from the phosphoenolpyruvate carboxykinase and apolipoprotein CIII genes also are effective in this setting. These results indicate that PKB/Akt functions downstream from PI3K in mediating sequence-specific effects of insulin on the expression of IGFBP-1 and perhaps multiple hepatic genes through a conserved IRS.

Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.

Curcumin has been shown to possess variety of biological functions including anti-tumor activity. The mechanism by which curcumin inhibit cell proliferation remains poorly understood. In the present report, we investigated the effect of curcumin on the activation of apoptotic pathway in T-cell acute lymphoblastic leukemia (T-ALL) malignant cells. Our data demonstrate that curcumin causes dose dependent suppression of proliferation in several T cell lines. Curcumin treatment causes the de-phosphorylation/inactivation of constitutively active AKT, FOXO transcription factor and GSK3. Curcumin also induces release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases, prevents caspase-3 activation and abrogates cell death induced by curcumin treatment. Finally, treatment of T-ALL cells with curcumin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Taken together, our finding suggest that curcumin suppresses constitutively activated targets of PI3′-kinase (AKT, FOXO and GSK3) in T cells leading to the inhibition of proliferation and induction of caspase-dependent apoptosis.

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells

p38α, p38β, p38γ, and p38δ are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38α and p38γ transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38δ mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38β is minimally expressed in early CD34+ hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38α, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38α, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38α does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38α and p38δ isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor α, an inflammatory cytokine that is modulated by p38α, is expressed by differentiating erythroblasts and inhibition of p38α or tumor necrosis factor α results in reduction in differentiation. Taken together, our data demonstrate that both p38α and δ isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Activation of Protein Kinase Cδ by IFN-γ

Engagement of the type II IFN (IFN-γ) receptor results in activation of the Janus kinase-Stat pathway and induction of gene transcription via IFN-γ-activated site (GAS) elements in the promoters of IFN-γ-inducible genes. An important event in IFN-γ-dependent gene transcription is phosphorylation of Stat1 on Ser727, which is regulated by a kinase activated downstream of the phosphatidylinositol 3′-kinase. Here we provide evidence that a member of the protein kinase C (PKC) family of proteins is activated downstream of the phosphatidylinositol 3′-kinase and is engaged in IFN-γ signaling. Our data demonstrate that PKCδ is rapidly phosphorylated during engagement of the type II IFNR and its kinase domain is induced. Subsequently, the activated PKCδ associates with a member of the Stat family of proteins, Stat1, which acts as a substrate for its kinase activity and undergoes phosphorylation on Ser727. Inhibition of PKCδ activity diminishes phosphorylation of Stat1 on Ser727 and IFN-γ-dependent transcriptional regulation via IFN-γ-activated site elements, without affecting the phosphorylation of the protein on Tyr701. Thus, PKCδ is activated during engagement of the IFN-γ receptor and plays an important role in IFN-γ signaling by mediating serine phosphorylation of Stat1 and facilitating transcription of IFN-γ-stimulated genes.

Activation of a CrkL-Stat5 Signaling Complex by Type I Interferons

Type I interferons (IFNα and IFNβ) transduce signals by inducing tyrosine phosphorylation of Jaks and Stats, as well as the CrkL adapter, an SH2/SH3-containing protein which provides a link to downstream pathways that mediate growth inhibition. We report that Stat5 interacts constitutively with the IFN receptor-associated Tyk-2 kinase, and during IFNα stimulation its tyrosine-phosphorylated form acts as a docking site for the SH2 domain of CrkL. CrkL and Stat5 then form a complex that translocates to the nucleus. This IFN-inducible CrkL-Stat5 complex binds in vitro to the TTCTAGGAA palindromic element found in the promoters of a subset of IFN-stimulated genes. Thus, during activation of the Type I IFN receptor, CrkL functions as a nuclear adapter protein and, in association with Stat5, regulates gene transcription through DNA binding.

Curcumin suppresses growth and induces apoptosis in primary effusion lymphoma

The mechanisms that regulate induction of the antiapoptotic state and mitogenic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we found that curcumin (diferuloylmethane), a natural compound isolated from the plant Curcuma Ionga, inhibits cell proliferation and induces apoptosis in a dose dependent manner in several PEL cell lines. Such effects of curcumin appear to result from suppression of the constitutively active STAT3 through inhibition of Janus kinase 1 (JAK1). Our data also demonstrate that curcumin induces loss of mitochondrial membrane potential with subsequent release of cytochrome c and activation of caspase-3, followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. Altogether, our findings suggest a novel function for curcumin, acting as a suppressor of JAK-1 and STAT3 activation in PEL cells, leading to inhibition of proliferation and induction of caspase-dependent apoptosis. Therefore, curcumin may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of STAT3.

Activation of Protein Kinase Cδ by All-trans-retinoic Acid

All-trans-retinoic acid (RA) is a potent inhibitor of leukemia cell proliferation and induces differentiation of acute promyelocytic leukemia cells in vitro and in vivo. For RA to induce its biological effects in target cells, binding to specific retinoic acid nuclear receptors is required. The resulting complexes bind to RA-responsive elements (RAREs) in the promoters of RA-inducible genes to initiate gene transcription and to generate protein products that mediate the biological effects of RA. In this report, we provide evidence that a member of the protein kinase C (PKC) family of proteins, PKCδ, is activated during RA treatment of the NB-4 and HL-60 acute myeloid leukemia cell lines as well as the MCF-7 breast cancer cell line. Such RA-dependent phosphorylation was also observed in primary acute promyelocytic leukemia cells and resulted in activation of the kinase domain of PKCδ. In studies aimed at understanding the functional relevance of PKCδ in the induction of RA responses, we found that pharmacological inhibition of PKCδ (but not of other PKC isoforms) diminished RA-dependent gene transcription via RAREs. On the other hand, overexpression of a constitutively active form of the kinase strongly enhanced RA-dependent gene transcription via RAREs. Gel shift assays and chromatin immunoprecipitation studies demonstrated that PKCδ associated with retinoic acid receptor-α and was present in an RA-inducible protein complex that bound to RAREs. Pharmacological inhibition of PKCδ activity abrogated the induction of cell differentiation and growth inhibition of NB-4 blast cells, demonstrating that its function is required for such effects. Altogether, our data provide strong evidence that PKCδ is activated in an RA-dependent manner and plays a critical role in the generation of the biological effects of RA in malignant cells.