Pleiotropic signaling evoked by tumor necrosis factor in podocytes.

TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-κB p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-κB inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-κB activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT)c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27(kip), or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La(3+)-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults.

STAT3 regulates steady-state expression of synaptopodin in cultured mouse podocytes.

The transcription factor signal transducer and activator of transcription-3 (STAT3) is activated by proinflammatory cytokines and circulating factors in many cell types. Synaptopodin (Synpo) is a cytoskeleton regulatory protein expressed in podocyte foot processes that regulates the dynamics of actin filaments and the stability of small GTPases. Here we show that inhibition of STAT3 signaling using the small-molecule inhibitor benzo[b]thiophene,6-nitro-,1,1-dioxide (Stattic), or by STAT3 knockdown by small interfering RNA, caused a decrease in Synpo mRNA and protein in an immortalized mouse podocyte cell line. This loss of Synpo, which occurred in 30-80 minutes, was also seen after treatment with the translational inhibitor cycloheximide. The loss of Synpo protein after Stattic or cycloheximide treatment did not occur when podocytes were simultaneously exposed to 1-[N-[(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl]amino]-4-guanidinobutane (E-64), an inhibitor of thiol proteases such as cathepsin L. Treatment with interleukin-6 (IL-6) increased tyrosine phosphorylation of STAT3 and evoked a parallel increase in Synpo levels in podocytes. The stimulatory effect of IL-6 on Synpo was completely inhibited by pretreatment with Stattic. By contrast, 30-60-minute exposure to angiotensin II (Ang II) inhibited STAT3 signaling and concurrently reduced Synpo protein levels. The Ang II-evoked loss of Synpo was prevented by E-64 but not by inhibition of calcineurin or blockade of transient receptor potential cation channels. Inhibition of STAT3 by Stattic caused marked changes in the distribution of podocyte actin filaments, and caused a nearly complete suppression of the migration of these cells in wound assays, consistent with the loss of Synpo. Stattic treatment also caused loss of RhoA protein.

Angiotensin II and canonical transient receptor potential-6 activation stimulate release of a signal transducer and activator of transcription 3-activating factor from mouse podocytes.

Previous studies have shown that the transcription factor signal transducer and activator of transcription-3 (STAT3) in podocytes plays an important role in progression of HIV nephropathy and in collapsing forms of glomerulonephritis. Here, we have observed that application of 100 nM angiotensin II (Ang II) to cultured podocytes for 6-24 hours causes a marked increase in the phosphorylation of STAT3 on tyrosine Y705 but has no effect on phosphorylation at serine S727. By contrast, Ang II treatment of short periods (20-60 minutes) caused a small but consistent suppression of tyrosine phosphylation of STAT3. A similar biphasic effect was seen after treatment with the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), an agent that causes activation of Ca(2+)-permeable canonical transient receptor potential-6 (TRPC6) channels in podocytes. The stimulatory effects of Ang II on STAT3 phosphorylation were abolished by small-interfering RNA knockdown of TRPC6 and also by inhibitors of the Ca(2+)-dependent downstream enzymes calcineurin and Ca(2+)-calmodulin-dependent protein kinase II. The stimulatory effects of Ang II appear to be mediated by secretion and accumulation of an unknown factor into the surrounding medium, as they are no longer detected when medium is replaced every 2 hours even if Ang II is continuously present. By contrast, the inhibitory effect of Ang II on STAT3 phosphorylation persists with frequent medium changes. Experiments with neutralizing and inhibitory antibodies suggest that the STAT3 stimulatory factor secreted from podocytes is not interleukin-6, but also suggest that this factor exerts its actions through a receptor system that requires glycoprotein 130.

The Time Course of JNK and P38 Activation in Cerebellar Granule Neurons Following Glucose Deprivation and BDNF Treatment.

Low glucose condition induces neuronal cell-death via intracellular mechanisms including mitogen-activated protein kinases (MAPK) signaling pathways. It has been shown that low glucose medium decreases neuronal survival in cerebellar granule neurons (CGNs). In this study, we have examined the activation of JNK, p38kinase and ERK1/2 pathways in low glucose medium in CGNs. The CGNs were prepared from new-born (P-2 and P-5) rats and cultured in Dulbecco's Modified Eagle's Medium high (DMEM-HIGH) glucose supplemented with Fetal Bovine Serum (FBS) 10% for 7 days. The glucose deprivation was induced through replacing the culture medium with the low glucose (5 mM) medium. The MAPK pathways activation was evaluated through phospho specific antibodies using western blot. The viability of cells was measuring using MTT assay. The results indicated that low glucose reduces the cell survival and brain-derived neurotrophic factor (BDNF) elevates the cell viability in CGNs. The basal c-Jun N-terminal kinase (JNK) activity was high in CGNs and glucose deprivation for 24 h had increased phospho-JNK level to 2-fold compared to basal. BDNF treatment reduced the basal JNK activity within 30 min but had no effect in longer incubations. BDNF also blocked the low glucose-induced JNK activation. In addition, CGNs exhibited high p38 phosphorylation in low glucose medium in 48 h. These results demonstrated that in sustained low glucose conditions, CGNs had high activity of stress-activated MAPK which could induce cellular damage. Moreover, BDNF can prevent JNK and p38 activation in stress conditions and increase cell viability. Our results suggest that in sustained stress conditions, inhibition of JNK and/or p38 pathways might protect neurons from damage in low glucose conditions.

The time course of Akt and ERK activation on XIAP expression in HEK 293 cell line.

The cell growth is controlled by the interaction of survival and cell growth arrest pathways as well as apoptosis mechanisms which determine the outcome of cell faith as proliferation or apoptosis. In this study, we have studied the activity of survival pathways, i.e., Akt and ERK1/2 with regard to XIAP (inhibitor of apoptosis) in serum starved and stimulated conditions. The HEK-293 cells were cultured in RPMI + 10% FBS. The cells were serum starved by switching to medium with 1% FBS for 24 h and serum stimulated by changing the medium to 10% FBS following serum starvation. The expression of p-Akt, p-ERK, Akt, ERK and XIAP was studied in various time points using western blot. The apoptosis was evaluated by DNA condensation using Hoechst 33258 and Caspase-3 assay. In serum starved condition expression of p-Akt and XIAP is very low. Serum stimulation increases p-Akt and p-ERK within 5 min and sustains a high level for 30 min. The expression of total Akt and ERK1/2 has not changed significantly for 24 h. XIAP expression starts at 6 h after serum stimulation, reaches to maximum level at 12 h and decreases to baseline within 24 h. Furthermore, serum starvation for 24 h does not induced apoptosis and DNA condensation. Taken together, the results indicate that serum activates Akt and ERK pathways earlier than XIAP expression. Furthermore, XIAP expression is low in serum starvation unlike p-ERK which suggests a survival role for ERK in serums starvation. The expression pattern of XIAP indicates induction by Akt and/or ERK activation which requires further studies.