Sengthong Chanchevalap

Krüppel-like factor 5 mediates the transforming activity of oncogenic H-Ras

Previous studies indicate that Krüppel-like factor 5 (KLF5), also known as intestinal-enriched Krüppel-like factor (IKLF), is a positive regulator of cell proliferation and gives rise to a transformed phenotype when overexpressed. Here we demonstrate that levels of KLF5 transcript and protein are significantly elevated in oncogenic H-Ras-transformed NIH3T3 cells. These cells display an accelerated rate of proliferation in both serum-containing and serum-deprived media and form anchorage-independent colonies in soft agar assays. H-Ras-transformed cells also contain elevated mitogen-activated protein kinase (MAPK) activity. When treated with inhibitors of MEK (MAPK kinase), H-Ras-transformed cells lose their growth advantage and no longer form colonies. Significantly, levels of KLF5 transcript and protein are substantially reduced in H-Ras-transformed cells treated with MEK inhibitors. Moreover, inhibition of KLF5 expression in H-Ras-transformed cells with KLF5-specific small interfering RNA (siRNA) leads to a decreased rate of proliferation and a significant reduction in colony formation. H-Ras-transformed cells also contain elevated levels of Egr1 that are diminished by MEK inhibitors. Inhibition of Egr1 by siRNA results in a reduced level of KLF5, indicating that Egr1 mediates the inductive action of MAPK on KLF5. Lastly, KLF5 activates expression of cyclin D1. These findings indicate that the increased expression of KLF5 in H-Ras-transformed cells is secondary to increased MAPK activity from H-Ras overexpression and that the elevated level of KLF5 is in part responsible for the proproliferative and transforming activities of oncogenic H-Ras.

Krüppel-like factor 5 is an important mediator for lipopolysaccharide-induced proinflammatory response in intestinal epithelial cells

Lipopolysaccharide (LPS) is a bacterially-derived endotoxin that elicits a strong proinflammatory response in intestinal epithelial cells. It is well established that LPS activates this response through NF-κB. In addition, LPS signals through the mitogen-activated protein kinase (MAPK) pathway. We previously demonstrated that the Krüppel-like factor 5 [KLF5; also known as intestine-enriched Krüppel-like factor (IKLF)] is activated by the MAPK. In the current study, we examined whether KLF5 mediates the signaling cascade elicited by LPS. Treatment of the intestinal epithelial cell line, IEC6, with LPS resulted in a dose- and time-dependent increase in KLF5 messenger RNA (mRNA) and protein levels. Concurrently, mRNA levels of the p50 and p65 subunits of NF-κB were increased by LPS treatment. Pretreatment with the MAPK inhibitor, U0126, or the LPS antagonist, polymyxin B, resulted in an attenuation of KLF5, p50 and p65 NF-κB subunit mRNA levels from LPS treatment. Importantly, suppression of KLF5 by small interfering RNA (siRNA) resulted in a reduction in p50 and p65 subunit mRNA levels and NF-κB DNA binding activity in response to LPS. LPS treatment also led to an increase in secretion of TNF-α and IL-6 from IEC6, both of which were reduced by siRNA inhibition of KLF5. In addition, intercellular adhesion molecule-1 (ICAM-1) levels were increased in LPS-treated IEC6 cells and this increase was associated with increased adhesion of Jurkat lymphocytes to IEC6. The induction of ICAM-1 expression and T cell adhesion to IEC6 by LPS were both abrogated by siRNA inhibition of KLF5. These results indicate that KLF5 is an important mediator for the proinflammatory response elicited by LPS in intestinal epithelial cells.

Effects of intra‐ and extracellular acidifications on single channel Kir2.3 currents

1 The inward rectifier K+ channel Kir2.3 is inhibited by hypercapnia, and this inhibition may be mediated by decreases in intra‐ and extracellular pH. To understand whether Kir2.3 has two distinct pH sensors and whether cytosol‐soluble factors are involved in the modulation of this channel during intracellular acidification, single channel currents were studied by expressing Kir2.3 in Xenopus oocytes. 2 In excised inside‐out patches, Kir2.3 currents had a high baseline channel open‐state probability (Po, at pH 7.4) with a strong inward rectification. Single channel conductance at hyperpolarizing membrane potential was about 17 pS with 150 mM K+ applied to both sides of the membrane. The channel showed a substate conductance of about 8 pS. 3 Reduction of intracellular pH (pHi) produced a fast and reversible inhibition of single channel Kir2.3 currents in inside‐out patches. The extent of this inhibition is concentration dependent. A clear reduction in Kir2.3 currents was seen at pHi 7.0, and channel activity was completely suppressed at pHi 6.2 with mid‐point inhibition (pK) at pH 6.77. 4 The effect of low pHi on Kir2.3 currents was due to a strong inhibition of Po and a moderate suppression of single channel conductance. The pK values for these single channel properties were pH 6.78 and 6.67, respectively. 5 The decrease in Po with low pHi resulted from an increase in the channel mean closed time without significant changes in the mean open time. Substate conductance was not seen during low pHi. 6 Decrease in extracellular pH (pHo) also caused inhibition of single channel activity of Kir2.3 currents in excised outside‐out patches. This effect, however, was clearly different from that of pHi: the pK (pH 6.70) was about 0.1 pH units lower; more than 50 % channel activity was retained at pHo 5.8; and low pHo affected mainly single channel conductance. 7 These results therefore indicate that (1) there are two distinct pH sensors in Kir2.3, (2) different mechanisms are involved in the modulation of Kir2.3 through these two pH sensors, and (3) cytosol‐soluble factors do not appear to be engaged in this modulation.

Lysophosphatidic acid facilitates proliferation of colon cancer cells via induction of Krüppel-like factor 5.

Among the multiple cellular effects mediated by lysophosphatidic acid (LPA), the effect on cell proliferation has extensively been investigated. A recent study showed that LPA-mediated proliferation of colon cancer cells requires activation of β-catenin. However, the majority of colon cancer cells have deregulation of the Wnt/β-catenin pathway. This prompted us to hypothesize the presence of additional pathway(s) activated by LPA resulting in an increase in the proliferation of colon cancer cells. Krüppel-like factor 5 (KLF5) is a transcriptional factor highly expressed in the crypt compartment of the intestinal epithelium. In this work, we investigated a role of KLF5 in LPA-mediated proliferation. We show that LPA stimulated the expression levels of KLF5 mRNA and protein in colon cancer cells and this stimulation was mediated by LPA2 and LPA3. Silencing of KLF5 expression by small interfering RNA significantly attenuated LPA-mediated proliferation of SW480 and HCT116 cells. LPA-mediated KLF5 induction was partially blocked by inhibition of the mitogen-activated protein kinase kinase and protein kinase C-δ. Moreover, we observed that LPA regulates KLF5 expression via eukaryotic elongation factor 2 kinase (eEF2k). Inhibition of calmodulin or silencing of eEF2k blocked the stimulation in KLF5 expression. Knockdown of eEF2k specifically inhibited KLF5 induction by LPA but not by fetal bovine serum or phorbol 12-myristate 13-acetate. These results identify KLF5 as a target of LPA-mediated signaling and suggest a role of KLF5 in promoting proliferation of intestinal epithelia in response to LPA.

Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons.

Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2.3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53.

Distinct Histidine Residues Control the Acid-induced Activation and Inhibition of the Cloned KATP Channel

The modulation of KATP channels during acidosis has an impact on vascular tone, myocardial rhythmicity, insulin secretion, and neuronal excitability. Our previous studies have shown that the cloned Kir6.2 is activated with mild acidification but inhibited with high acidity. The activation relies on His-175, whereas the molecular basis for the inhibition remains unclear. To elucidate whether the His-175 is indeed the protonation site and what other structures are responsible for the pH-induced inhibition, we performed these studies. Our data showed that the His-175 is the only proton sensor whose protonation is required for the channel activation by acidic pH. In contrast, the channel inhibition at extremely low pH depended on several other histidine residues including His-186, His-193, and His-216. Thus, proton has both stimulatory and inhibitory effects on the Kir6.2 channels, which attribute to two sets of histidine residues in the C terminus.

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains.

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO2/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO2 and pH i . These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K+ channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.

CO2 inhibits specific inward rectifier K+ channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K+ channels. To understand the mechanisms for this modulation, four cloned K+ channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO2 for 4–6 min produced reversible and concentration‐dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra‐ and extracellular pH (pHi, pHo) dropped during CO2 exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra‐ and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell‐free excised inside‐out patches with cytosolic‐soluble factors washed out, a decrease in pHi produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pHi level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO2 without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K+ channels, (2) these inhibitions are caused by intra‐ and extracellular protons rather than molecular CO2, and (3) these effects are independent of cytosol‐soluble factors. J. Cell. Physiol. 183:53–64, 2000.

A single residue contributes to the difference between Kir4.1 and Kir1.1 channels in pH sensitivity, rectification and single channel conductance

1 Kir1.1 and Kir4.1 channels may be involved in the maintenance of pH and K+ homeostasis in renal epithelial cells and CO2 chemoreception in brainstem neurons. To understand the molecular determinants for their characteristic differences, the structure‐function relationship was studied using site‐directed mutagenesis. 2 According to previous studies, Glu158 in Kir4.1 is likely to be the major rectification controller. This was confirmed in both Kir1.1 and Kir4.1. Mutation of Gly210, the second potential rectification controller, to glutamate did not show any additional effect on the inward rectification. 3 More interestingly, we found that Glu158 in Kir4.1 was also an important residue contributing to single channel conductance and pH sensitivity. The E158N Kir4.1 mutant had a unitary conductance of 35 pS and a midpoint pH for channel inhibition (pKa) value of 6.72, both of which were almost identical to those of the wild‐type (WT) Kir1.1. Flickering channel activity was clearly seen in the E158N mutant at positive membrane potentials, which is typical in the WT Kir1.1 but absent in the WT Kir4.1. 4 Reverse mutation in Kir1.1 (N171E) reduced the unitary conductance to 27 pS (23 pS in WT Kir4.1). However, the pH sensitivity of this mutant did not show a marked difference from the WT Kir1.1. Therefore, it is possible that a residue(s) in addition to Asn171 is also involved. Thus we studied several other residues in both M2 and H5 regions. We found that joint mutations of Val140 and Asn171 to residues seen in Kir4.1 greatly reduced the pH sensitivity (pKa 6.08). 5 The V140T mutation in Kir1.1 led to a unitary conductance of ∼70 pS, and the G210E mutation in Kir4.1 caused a decrease in pH sensitivity of 0.4 pH units. 6 These results indicate that the pore‐forming sequences are targets for modulations of multiple channel‐biophysical properties and demonstrate a site contributing to rectification, unitary conductance and proton sensitivity in these Kir channels.