Cyclin D1 activation through ATF-2 in Reg-induced pancreatic beta-cell regeneration

Regenerating gene product (Reg) is induced in pancreatic beta-cells and acts as an autocrine/paracrine growth factor for regeneration via a cell surface Reg receptor. However, the manner by which Reg induces beta-cell regeneration was unknown. In the present study, we found that Reg increased phospho-ATF-2, which binds to -57 to -52 of the cyclin D1 gene to activate the promoter. The Reg/ATF-2-induced cyclin D1 promoter activation was attenuated by PI(3)K inhibitors such as LY294002 and wortmannin. In Reg knockout mouse islets, the levels of phospho-ATF-2, cyclin D1, and phospho-Rb were greatly decreased. These results indicate that the Reg-Reg receptor system stimulates the PI(3)K/ATF-2/cyclin D1 signaling pathway to induce beta-cell regeneration.     

Analysis of the mechanism regulating the stability of rat macrophage inflammatory protein-2 mRNA in RBL-2H3 cells

Using rat peritoneal neutrophils, the complete nucleotide sequence of rat macrophage inflammatory protein-2 (MIP-2) mRNA including 5'untranslated region (UTR) and 3'UTR was determined (GenBank Accession number, AB060092). It was found that the MIP-2 mRNA has a 70 bp 5'UTR, a 303 bp coding region and a 728 bp 3'UTR which contains adenylate/uridylate (AU)-rich areas defined as AU-rich elements (AREs). Site-directed mutagenesis studies using the tetracycline-sensitive transactivator protein-expressing rat basophilic leukemia cells (RBL-2H3-TO cells) revealed that MIP-2 mRNA mutants which lack the 3'UTR are more stable than MIP-2-wild-type (wt) mRNA. A MIP-2 mRNA mutant in which some mutations were introduced to the ARE was also stable. The stability of MIP-2 mRNA was low in untreated RBL-2H3-TO cells, but it increased in the antigen-stimulated immunoglobulin E (IgE)-sensitized cells. The antigen-induced MIP-2 mRNA stabilization was counteracted by the highly specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD98059. These findings indicate that ARE is the cis-element which mediates the rapid decay of MIP-2 mRNA, and the antigen stimulation stabilizes MIP-2 mRNA and the p38 MAPK and p44/42 MAPK pathways are involved in the antigen-induced stabilization of MIP-2 mRNA.     

ATF-2 cooperates with Smad3 to mediate TGF-beta effects on chondrocyte maturation

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-beta. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-beta, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-beta-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-beta. However, in combination the effects were additive and similar to the inhibitory effects of TGF-beta on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-beta on colX, while together the blockade was complete. Similar effects were observed with another TGF-beta-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-beta signaling. In contrast, although TGF-beta does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-beta and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-beta signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-beta or BMP-2.     

The MEF2A and MEF2D function as scaffold proteins that interact with HDAC1 or p300 in SOD3 expression in THP-1 cells

Superoxide dismutase 3 (SOD3) is a SOD isozyme and plays a key role in extracellular redox homeostasis. We previously demonstrated that histone acetylation is involved in 12-O-tetra-decanoylphorbol-13-acetate (TPA)-elicited SOD3 expression in human monocytic THP-1 cells; however, the molecular mechanisms responsible for its expression have not yet been elucidated in detail. The results of the present study demonstrated that the binding of histone deacetylase 1 (HDAC1) to the SOD3 promoter region contributed to SOD3 silencing in basal THP-1 cells. On the other hand, the dissociation of HDAC1 from the SOD3 promoter region and the enrichment of p300, a histone acetyltransferase (HAT), within that region were observed in TPA-induced THP-1 cells. Myocyte enhancer factor 2 (MEF2) functions as a scaffold protein that interacts with histone deacetylases (HDAC) or HAT and regulates gene expression. The present results showed that the MEF2A and MEF2D function as mediators for TPA-elicited SOD3 expression by interacting with HDAC or p300. Additionally, the knockdown of MEF2A or MEF2D in human skin fibroblasts suppressed SOD3 expression at the mRNA and protein levels. Our results provide an insight into epigenetic regulation of redox gene expression, and may ultimately contribute to suppressing the progression of tumours and vascular diseases.     

Murine mammary carcinoma cells and CD11c dendritic cells elicit distinct responses to lipopolysaccharide and exhibit differential expression of genes required for TLR4 signaling

Although TLR are often studied on DC because of their ability to bridge innate and adaptive defenses, TLR are also expressed by epithelial cells. Because the majority of cancers are carcinomas, and thus of epithelial origin, we wanted to know whether a carcinoma and DC responded similarly to a TLR agonist. We found the mammary carcinoma 4T1 and CD11c⁺ DC both secreted proinflammatory chemokines in response to the TLR4 agonist lipopolysaccharide (LPS). However a clear dichotomy existed. DC, but not 4T1 secreted IL-1β, TNF-α, and upregulated CD80 and CD86 expression following LPS treatment. A potential reason for differential responsiveness was that DC expressed greater levels of TLR4, CD14, Myd88, and TRAM. Despite the low level of TLR signaling proteins, the carcinoma were able to elicit a range of responses contingent upon the source, dose, length, and frequency of TLR agonist treatment. Thus, carcinoma and DC are distinctly responsive to LPS.     

Inhibition of RIP2/RICK/CARDIAK activity by pyridinyl imidazole inhibitors of p38 MAPK

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase (MAPK) have been used extensively in vitro and in vivo to investigate the role of p38 in physiological processes. As with other pharmacological inhibitors, non-specific targets of the p38 inhibitors have been reported. We have found that the protein kinase receptor interacting protein-2 (RIP2) is another target for the family of p38 inhibitors. The autophosphorylation of RIP2 was inhibited in vitro by the p38 inhibitors SB220025, SB203580 and PD169316 at concentrations comparable to those used to inhibit p38. We also identified two new in vitro substrates for RIP2, myelin basic protein and histone H3 with apparent K_m values of 2.1 M and 0.65 M, respectively. The ability of RIP2 to phosphorylate these two substrates was sensitive to the p38 inhibitors as well. As was shown for p38, a conserved threonine in the kinase domain of RIP2 is required for sensitivity to the inhibitors, indicating that the mechanism of inhibition of RIP2 is similar to that of p38. These results demonstrate that the pyridinyl imidazole inhibitors block RIP2 as well as p38 kinase activity. (Mol Cell Biochem 268: 129–140, 2005)     

Zinc differentially regulates mitogen-activated protein kinases in human T cells

Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.     

Differentiation-inducing potency of the seco-steroid JK-1624F2-2 can be increased by combination with an antioxidant and a p38MAPK inhibitor which upregulates the JNK pathway

Low calcemic analogs of vitamin D are candidates for differentiation therapy of human myeloid leukemias. We report here that the seco-steroid synthesized to have resistance to intracellular degradation and low calcemia-inducing activity, 1alpha-hydroxymethyl-3beta-16-ene-24,24-difluoro-25-hydroxy-vitamin D₃ (JKF), induces monocytic differentiation in four established human myeloid leukemia cell lines, HL60, U937, THP-1, NB-4, and murine myeloid leukemia cells WEHI-3B D⁻. JKF has differentiation-inducing potency which is slightly lower than the physiologically active form of vitamin D, 1,25(OH)₂vitamin D₃ (1,25D). However, simultaneous addition of carnosic acid (CA), an antioxidant, and SB20190 (SB), an inhibitor of p38MAP kinase, increases the differentiation efficiency of JKF to a level similar to the level observed when 1,25D is used in such combinations. We also show for the first time that SB inhibits the phosphorylation of MAPKAPK2, a downstream target of p38MAPK, but upregulates the phosphorylation of at least one of the isoforms of JNK (p46 JNK1) and of c-jun in all four human myeloid cell lines studied here. These studies indicate that the JNK1 pathway is positively associated with monocytic differentiation of several subtypes of myeloid leukemia cells arrested at different developmental stages. Further, since JKF is less calcemic than 1,25D, the data suggest that JKF combined with CA and SB is likely to have a therapeutic advantage over 1,25D-based experimental regimens for myeloid leukemias.