Tumor necrosis factor α (TNF-α) induces the expression and secretion of interleukin 8 (IL-8), which contributes to synovitis in rheumatoid arthritis (RA). To elucidate the mechanism of the onset of RA, we used synovial fibroblasts without autoimmune inflammatory diseases and investigated MAPK signaling pathways in TNF-α-induced IL-8 expression. Synovial fibroblasts isolated from healthy dogs were characterized by flow cytometry, which were positive for the fibroblast markers CD29, CD44, and CD90 but negative for the hematopoietic cell markers CD14, CD34, CD45, and HLA-DR. TNF-α stimulated the secretion and mRNA expression of IL-8 in a time- and dose-dependent manner. ERK and JNK inhibitors attenuated TNF-α-induced IL-8 expression and secretion. TNF-α induced the phosphorylation of ERK1/2 and JNK1/2. TNF-α-induced IL-8 expression was attenuated both in ERK2- and JNK1-knockdown cells. TNF-α-induced ERK1/2 or JNK1/2 was observed in ERK2- or JNK1-knockdown cells, respectively, showing that there is no crosstalk between ERK2 and JNK1 pathways. These observations indicate that the individual activation of ERK2 and JNK1 pathways contributes to TNF-α-induced IL-8 expression in synovial fibroblasts, which appears to be involved in the progress in RA.

The proinflammatory cytokine interleukin 1β (IL-1β) induces prostaglandin E₂ (PGE₂) production via upregulation of cyclooxygenase-2 (COX-2) expression in synovial fibroblasts. This effect of IL-1β is involved in osteoarthritis. We investigated MAPK signaling pathways in IL-1β-induced COX-2 expression in feline synovial fibroblasts. In the presence of MAPK inhibitors, IL-1β-induced COX-2 expression and PGE₂ release were both attenuated. IL-1β induced the phosphorylation of p38, JNK, MEK, and ERK1/2. A JNK inhibitor prevented not only JNK phosphorylation but also MEK and ERK1/2 phosphorylation in IL-1β-stimulated cells, but MEK and ERK1/2 inhibitors had no effect on JNK phosphorylation. A p38 inhibitor prevented p38 phosphorylation, but had no effect on MEK, ERK1/2, and JNK phosphorylation. MEK, ERK1/2, and JNK inhibitors had no effect on p38 phosphorylation. We also observed that in IL-1β-treated cells, phosphorylated MEK, ERK1/2, and JNK were co-precipitated with anti-phospho-MEK, ERK1/2, and JNK antibodies. The silencing of JNK1 in siRNA-transfected fibroblasts prevented IL-1β to induce phosphorylation of MEK and ERK1/2 and COX-2 mRNA expression. These observations suggest that JNK1 phosphorylation is necessary for the activation of the MEK/ERK1/2 pathway and the subsequent COX-2 expression for PGE₂ release, and p38 independently contributes to the IL-1β effect in synovial fibroblasts.

JNK is a protein kinase, which induces transactivation of c-jun. The three isoforms of JNK, JNK1, JNK2, and JNK3, are encoded by three distinct genes. JNK1 and JNK2 are expressed ubiquitously throughout the body. By contrast, the expression of JNK3 is limited and observed mainly in the brain, heart, and testes. Concerning the biological properties of JNKs, the contribution of upstream regulators and scaffold proteins plays an important role in the activation of JNKs. Since JNK signaling has been described as a form of stress-response signaling, the contribution of JNK3 to pathophysiological events, such as stress response or cell death including apoptosis, has been well studied. However, JNK3 also regulates the physiological functions of neurons and non-neuronal cells, such as development, regeneration, and differentiation/reprogramming. In this review, we shed light on the physiological functions of JNK3. In addition, we summarize recent advances in the knowledge regarding interactions between JNK3 and cellular reprogramming.

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca(2+) mobilization of the cells was evaluated using the Ca(2+) indicator Fluo3 to monitor Ca(2+) influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca(2+) levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.

Acute kidney injury (AKI) is characterized by a sudden loss of renal function. Early recognition of AKI, especially in critically ill patients, is essential for adequate therapy. Currently, neutrophil gelatinase-associated lipocalin (NGAL) is considered to be an effective biomarker of AKI; however, the regulation of its expression and function in renal tubular cells remains unclear. In this study, we investigated the regulation of the expression and function of NGAL in IL-1β-treated Madin–Darby canine kidney (MDCK) cells as a model of renal tubular cells. IL-1β induced a disturbance in the localization of E-cadherin and zonaoccludin-1 (ZO-1). The transepithelial electrical resistance (TER) also decreased 5 days after IL-1β treatment. IL-1β induced NGAL mRNA expression and protein secretion in a time- and dose-dependent manner, which occurred faster than the decrease in TER. In the presence of ERK1/2 and p38 inhibitors, IL-1β-induced NGAL mRNA expression and protein secretion were significantly attenuated. In the presence of recombinant NGAL, IL-1β-induced disturbance in the localization of E-cadherin and ZO-1 was attenuated, and the decrease in TER was partially maintained. These results suggest that NGAL can be used as a biomarker for AKI and that it functions as a protector from AKI.