FOXA1 and IRF-1 intermediary transcriptional regulators of PPARgamma-induced urothelial cytodifferentiation.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that has been implicated in the induction of differentiation of various cell types, including human uroepithelial cells. PPARgamma-mediated differentiation of normal human urothelial (NHU) cells in vitro requires coinhibition of epidermal growth factor receptor (EGFR) signalling and is characterised by de novo expression of late/terminal differentiation-associated genes, including uroplakins (UPK), over a 6-day period. We used gene microarrays to identify intermediary transcription factors induced in direct response to PPARgamma activation of EGFR-inhibited NHU cells. FOXA1 and IRF-1 contained consensus cognate binding sites in UPK1a, UPK2, and UPK3a promoters and transcripts were induced within 12 h of PPARgamma activation; transcription complex formation was confirmed by electromobility shift assays. In urothelium in situ, both FOXA1 and IRF-1 were nuclear and expressed in a differentiation-associated pattern. Knockdown by transient siRNA of either FOXA1 or IRF-1 abrogated PPARgamma-induced uroplakin expression in vitro. This is the first evidence that ligand activation of PPARgamma induces expression of intermediary transcription factors that mediate an epithelial differentiation programme and represents a new paradigm for understanding differentiation, regenerative repair and inflammation in epithelial tissues.

Modelling cell signalling and differentiation in the urothelium.

Urothelial cells line the bladder. If the urothelium is damaged, it is vital that it repairs itself quickly. Experimental results shedding light on how this repair process works are presented, revealing in particular the dependence of the response on the length of time for which the drug Troglitazone (TZ) is applied. A simple mathematical model for the basic mechanism (comprising ordinary differential equations) is then developed and analysed, seeking specifically to clarify and quantify the mechanisms governing the dependence of the cell differentiation response on the TZ administration time, rather than providing a comprehensive model of differentiation. Through biologically justified simplifications, analysis reveals that the model gives results in accord with the experimental observations, and suggests new experiments that may aid further understanding. Directions in which this preliminary modelling of the PPAR gamma (peroxisome proliferator activated receptor gamma) pathway could be usefully extended are also indicated.

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway.

Hepatocyte isolation stimulates formation of interferon stimulatory response element DNA-protein complexes.

We have examined the relationship between intracellular signalling pathways and loss of differentiated function during hepatocyte isolation and culture. We have shown that isolation induces the activation of the interferon stimulatory response element (ISRE). This activation was transient and peaked at 3 h before it returned to basal by 24 h of culture. Interferon regulatory factor-1 (IRF-1) was shown to be important for generation of ISRE complexes by electromobility shift assays and supershift intervention. IRF-1 was translocated to the nucleus in parallel with changes to ISRE complex formation. The p38 kinase inhibitor, SB 203580, diminished the formation of ISRE binding complexes. Hence p38 kinase may be involved in the activation and binding of IRF-1 or related proteins to the ISRE motif. Changes in ISRE activation levels in cultured hepatocytes may have important implications in primary hepatocyte differentiation and loss of function.

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression.

Gene 33 is a putative immediate early gene and we have shown that mRNA encoding for gene 33 exhibits a transient increase as a result of the procedures used for hepatocyte isolation. The stress-activated protein kinases p46 JNK, p54 JNK, and p38 SAPK are activated by hepatocyte isolation and precede changes in gene 33 mRNA content. Although each SAPK isoform shows a distinctive profile of activity during isolation and subsequent hepatocyte culture, in each case the activation is transient and is largely reversed within 3 h of hepatocyte isolation. SB 203580, a p38 SAPK inhibitor, prevents the change to gene 33 expression in response to hepatocyte isolation. Given the possible role of gene 33 as an immediate early gene, the data presented here have general implications for control of hepatocyte proliferation and differentiation.

Interleukin-1beta-induced expression of protein kinase C (PKC)-delta and epsilon in NIH 3T3 cells.

NIH 3T3 cells express the alpha, delta, epsilon and zeta isoenzymes of protein kinase C(PKC). Following stimulation of cells (24 h) with the pro-inflammatory cytokine, interleukin 1beta (IL-1beta), we observed, by Western blotting, a dose-dependent effect on the levels of PKC-epsilon and delta, but not on alpha or zeta. Moreover, time course analysis revealed that the isoenzymes, PKC-delta and epsilon were induced by IL-1beta after 7 h. Again, no change in PKC-alpha or zeta levels after IL-1beta treatment were detected. Incubation with selective PKC inhibitor peptides blocked the PKC-alpha, delta, epsilon and zeta antibodies binding to their respective isoenzyme bands. We also observed that the addition of the tumour-promoting phorbol ester, Phorbol 12-myristate 13-acetate (PMA), downregulated PKC-alpha, delta and epsilon by 7 h in NIH 3T3 cells. PMA did not affect constitutively produced PKC-zeta protein levels even after 24-h treatment. In summary, these results demonstrate that IL-1beta induces protein synthesis of the Ca2+-independent PKC-delta and epsilon isoforms in NIH 3T3 cells. The differences observed here between PKC isoenzymes in response to IL-1beta suggest that each isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such selective expression may influence the action of agents which require PKC for signal transduction acting in concert with IL-1.

Characterization of inositol monophosphatase in human cerebrospinal fluid.

Inositol monophosphatase (IMPase) has been identified and characterized in human lumbar cerebrospinal fluid (CSF). The CSF enzyme has a Km for inositol 1-phosphate (Ins(1)P; 0.12 mM), a magnesium dependence (optimum concentration 10 mM) and a sensitivity to inhibition by either the bisphosphonate inhibitor 1-(4-hydroxyphenyloxy)ethane-1,1-bisphosphonic acid (L-690,330) or LiCl (IC50's: 1.3 microM and 1.6 mM, respectively) similar to native human brain and human recombinant enzymes. In CSF, antiserum raised against purified bovine brain IMPase recognised a protein of 30 kDa, identical to that seen in human brain homogenate. It remains to be determined whether CSF IMPase activity may be a useful in vivo marker of CNS phosphatidyl inositol cycle activity in disorders where this signalling pathway may be altered (e.g. manic depression).