Sung‐Ho Huh

NO, produced from l-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, we demonstrate that endogenously produced NO can suppress c-Jun N-terminal kinase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-gamma induced endogenous NO production, concomitantly suppressing JNK1 activation. Similarly, IFN-gamma induced suppression of JNK1 activation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-gamma-induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented by N(G)-nitro-l-arginine, a NO synthase inhibitor. Interestingly, the IFN-gamma-induced suppression of JNK1 activation was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-permeant analogue of cGMP, did not change JNK1 activation in intact cells either. In contrast, S-nitro-N-acetyl-dl-penicillamine (SNAP), a NO donor, inhibited JNK1 activity in vitro. Furthermore, a thiol reducing agent, DTT, reversed not only the in vitro inhibition of JNK1 activity by SNAP but also the in vivo suppression of JNK1 activity by IFN-gamma. Substitution of serine for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-gamma or SNAP on JNK1 activity in vivo or in vitro, respectively. Moreover, IFN-gamma enhanced endogenous S-nitrosylation of JNK1 in RAW264.7 cells. Collectively, our data suggest that endogenous NO mediates the IFN-gamma-induced suppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.

Heat shock protein 72 (Hsp72) is thought to protect cells against cellular stress. The protective role of Hsp72 was investigated by determining the effect of this protein on the stress-activated protein kinase signaling pathways. Prior exposure of NIH 3T3 cells to mild heat shock (43 degrees C for 20 min) resulted in inhibition of H(2)O(2)-induced activation of apoptosis signal-regulating kinase 1 (ASK1). Overexpression of Hsp72 also inhibited H(2)O(2)-induced activation of ASK1 as well as that of downstream kinases in the p38 mitogen-activated protein kinase (MAPK) signaling cascade. Recombinant Hsp72 bound directly to ASK1 and inhibited ASK1 activity in vitro. Furthermore, coimmunoprecipitation analysis revealed a physical interaction between endogenous Hsp72 and ASK1 in NIH 3T3 cells exposed to mild heat shock. Hsp72 blocked both the homo-oligomerization of ASK1 and ASK1-dependent apoptosis. Hsp72 antisense oligonucleotides prevented the inhibitory effects of mild heat shock on H(2)O(2)-induced ASK1 activation and apoptosis. These observations suggest that Hsp72 functions as an endogenous inhibitor of ASK1.

In hair follicle development, a placode-derived signal is believed to induce formation of the dermal condensation, an essential component of ectodermal organs. However, the identity of this signal is unknown. Furthermore, although induction and patterning of hair follicles are intimately linked, it is not known whether the mesenchymal condensation is necessary for inducing the initial epithelial pattern. Here, we show that fibroblast growth factor 20 (Fgf20) is expressed in hair placodes and is induced by and functions downstream from epithelial ectodysplasin (Eda)/Edar and Wnt/β-Catenin signaling to initiate formation of the underlying dermal condensation. Fgf20 governs formation of primary and secondary dermal condensations in developing hair follicles and subsequent formation of guard, awl, and auchene hairs. Although primary dermal condensations are absent in Fgf20 mutant mice, a regular array of hair placodes is formed, demonstrating that the epithelial patterning process is independent of known histological and molecular markers of underlying mesenchymal patterns during the initial stages of hair follicle development.

The protein serine-threonine kinase Akt mediates cell survival signaling initiated by various growth-promoting factors such as insulin. Here we report that SEK1 is a target of Akt in intact cells. Insulin inhibited the anisomycin-induced stimulation of both endogenous SEK1 and its substrate c-Jun N-terminal kinase (JNK), but not that of the upstream kinase MEKK1, in 293T cells. The inhibitory action of insulin on SEK1 or JNK1 activation was prevented by the phosphatidylinositol 3-kinase inhibitor LY294002. Expression of a constitutively active form of Akt also inhibited both SEK1 and JNK1 activation, but not that of MEKK1, in transfected 293T cells. Co-immunoprecipitation analysis revealed that endogenous Akt physically interacted with endogenous SEK1 in cells and that this interaction was promoted by insulin. In vitro and in vivo (32)P labeling indicated that Akt phosphorylated SEK1 on serine 78. The SEK1 mutant SEK1(S78A) was resistant to Akt-induced inhibition. Finally, activated Akt inhibited SEK1-mediated apoptosis, and this effect of Akt was prevented by overexpression of SEK(S78A). Taken together, these results suggest that Akt suppresses stress-activated signaling by targeting SEK1.