Markedly attenuated acute and chronic pain responses in mice lacking adenylyl cyclase-5.

Chronic inflammatory and neuropathic pain is often difficult to manage using conventional remedies. The underlying mechanisms and therapeutic strategies required for the management of chronic pain need to be urgently established. The cyclic AMP (cAMP) second messenger system has been implicated in the mechanism of nociception, and the inhibition of the cAMP pathway by blocking the activities of adenylyl cyclase (AC) and protein kinase A has been found to prevent chronic pain in animal models. However, little is known regarding which of the 10 known isoforms of AC are involved in nociceptive pathways. Therefore, we investigated the potential pronociceptive function of AC5 in nociception using recently developed AC5 knockout mice (AC5-/-). We found that AC5-/- mice show markedly attenuated pain-like responses in acute thermal and mechanical pain tests as compared with the wildtype control. Also, AC5-/- mice display hypoalgesic responses to inflammatory pain induced by subcutaneous formalin injection into hindpaws, and to non-inflammatory and inflammatory visceral pain induced by injecting magnesium sulfate or acetic acid into the abdomen. Moreover, AC5-/- mice show strongly suppressed mechanical and thermal allodynia in two nerve injury-induced neuropathic pain models. These results suggest that AC5 is essential for acute and chronic pain, and that AC5 knockout mice provide a useful model for the evaluation of the pathophysiological mechanisms of pain.

Molecular identification of IgE-dependent histamine-releasing factor as a B cell growth factor.

The culture supernatants of LK1 cells, murine erythroleukemia cells, showed B cell-stimulating activity. Purification and NH(2)-terminal sequence analysis revealed that one of the candidates was murine IgE-dependent histamine-releasing factor (IgE-HRF), which is known to induce histamine from basophils. Recombinant IgE-HRF (rHRF) obtained from Escherichia coli- or 293-transformed embryonal kidney cells was tested for B cell-stimulating activity. Both rHRFs stimulated B cell proliferation in a dose-dependent manner. However, boiling or anti-HRF Ab abolished the B cell stimulatory effects of rHRF. Recombinant HRF showed strong synergistic effects with IL-2, IL-4, and IL-5 for B cell activation, with maximal activity in the presence of anti-CD40 AB: Recombinant HRF increased MHC class II expression of B cells. It also increased Ig production from B cells. Treatment with polymyxin B, a neutralizing peptide antibiotic of LPS, did not reduce the activity of rHRF. In addition, FACS analysis using PE-conjugated rHRF showed that HRF bound to B cells. Recombinant HRF up-regulated the expression of IL-1 and IL-6 in B cells. In vivo administration of rHRF or the cDNA for rHRF increased total and Ag-specific Ig synthesis. Taken together, these results indicate that HRF stimulates B cell activation and function.

IFN-gamma up-regulates IL-18 gene expression via IFN consensus sequence-binding protein and activator protein-1 elements in macrophages.

Constitutive IL-18 expression is detected from many different cells, including macrophages, keratinocytes, and osteoblasts. It has been known that IL-18 gene expression is regulated by two different promoters (p1 promoter and p2 promoter). When RAW 264.7 macrophages were treated with IFN-gamma, IL-18 gene expression was increased in a dose- and time-dependent manner. IFN-gamma activated the inducible promoter 1, but not the constitutive promoter 2. Mutagenesis studies indicated that an IFN consensus sequence-binding protein (ICSBP) binding site between -39 and -22 was critical for the IFN-gamma inducibility. EMSA using an ICSBP oligonucleotide probe showed that IFN-gamma treatment increased the formation of DNA-binding complex, which was supershifted with anti-IFN regulatory factor-1 Ab and anti-ICSBP Ab. Another element, an AP-1 site between -1120 and -1083, was important. EMSA using an AP-1-specific oligonucleotide demonstrated that IFN-gamma or LPS treatment increased the AP-1-binding activity. The addition of anti-c-Jun Ab or anti-c-Fos Ab to IFN-gamma- or LPS-treated nuclear extracts resulted in the reduction of AP-1 complex or the formation of a supershifted complex. Taken together, these results indicate that IFN-gamma increased IL-18 gene expression via ICSBP and AP-1 elements.

Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression.

Stimulation of human lung fibroblast cells with TGF-beta1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l -cysteine (NAC). TGF-beta1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-beta1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-beta1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-beta1 treatment. EGTA suppressed TGF-beta1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-beta1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-beta1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-beta1-induced IL-6 expression. Taken together, these results indicate that TGF-beta1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function.

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.