Masahiro Matsubara

A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC → YojN → RcsB signalling pathway implicated in capsular synthesis and swarming behaviour

In this study, we re‐investigated the previously characterized RcsC (sensor His‐kinase) → RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His → Asp phosphorelay, this RcsC → RcsB framework is somewhat puzzling, because RcsC appears to contain both a His‐kinase domain and a receiver domain, but not a histidine (His)‐containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His → Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His‐containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC → YojN → RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.

Tuning of the porin expression under anaerobic growth conditions by His-to-Asp cross-phosphorelay through both the EnvZ-osmosensor and ArcB-anaerosensor in Escherichia coli

Widespread bacterial signal transduction circuits are generally referred to as ‘two‐component systems’ or ‘histidine (His)‐to‐aspartate (Asp) phosphorelays.’ In Escherichia coli, as many as 30 distinct His‐to‐Asp phosphorelay signalling pathways operate in response to a wide variety of environmental stimuli, such as medium osmolarity and anaerobiosis. In this regard, it is of interest whether or not some of them together constitute a network of signalling pathways through a physiologically relevant mechanism (often referred to as ‘cross‐regulation’). We have addressed this issue, with special reference to the osmo‐responsive EnvZ and anaero‐responsive ArcB phosphorelay signalling pathways in E. coli.

An Escherichia coli protein that exhibits phosphohistidine phosphatase activity towards the HPt domain of the ArcB sensor involved in the multistep His-Asp phosphorelay.

The Escherichia coli sensory kinase, ArcB, possesses a histidine‐containing phosphotransfer (HPt) domain, which is implicated in the His‐Asp multistep phosphorelay. We searched for a presumed phosphohistidine phosphatase, if present, which affects the function of the HPt domain through its dephosphorylation activity. Using in vivo screening, we first identified a gene that appeared to interfere with the His‐Asp phosphorelay between the HPt domain and the receiver domain of OmpR, provided that the gene product was expressed through a multicopy plasmid. The cloned gene (named sixA) was found to encode a protein consisting of 161 amino acids, which has a noticeable sequence motif, an arginine–histidine–glycine (RHG) signature, at its N‐terminus. Such an RHG signature, which presumably functions as a nucleophilic phosphoacceptor, was previously found in a set of divergent enzymes, including eukaryotic fructose‐2,6‐bisphosphatase, E. coli periplasmic phosphatase and E. coli glucose‐1‐phosphate phosphatase, and ubiquitous phosphoglycerate mutase. Otherwise, the entire amino acid sequences of none of these enzymes resembles that of SixA. It was demonstrated in vitro that the purified SixA protein exhibited the ability to release the phosphoryl group from the HPt domain of ArcB, but the mutant protein lacking the crucial histidine residue in the RHG signature did not. Evidence was also provided that a deletion mutation of sixA on the chromosome affected the in vivo phosphotransfer signalling. These results support the view that SixA is capable of functioning as a phosphohistidine phosphatase that may be implicated in the His‐Asp phosphorelay through regulating the phosphorylation state of the HPt domain.

Blockade of T-type voltage-dependent Ca2+ channels by benidipine, a dihydropyridine calcium channel blocker, inhibits aldosterone production in human adrenocortical cell line NCI-H295R.

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels.

The SixA phospho-histidine phosphatase modulates the ArcB phosphorelay signal transduction in Escherichia coli

The Escherichia coli SixA protein is the first discovered prokaryotic phospho‐histidine phosphatase, which was implicated in a His‐to‐Asp phosphorelay. The sixA gene was originally identified as the one that interferes with, at its multi‐copy state, the cross‐phosphorelay between the histidine‐containing phosphotransmitter (HPt) domain of the ArcB anaerobic sensor and its non‐cognate OmpR response regulator. Nevertheless, no evidence has been provided that the SixA phosphatase is indeed involved in a signaling circuitry of the authentic ArcB‐to‐ArcA phosphorelay in a physiologically meaningful manner. In this study, a SixA‐deficient mutant was characterized with special reference to the ArcB signaling, which allows E. coli cells to respond to not only external oxygen, but also certain anaerobic respiratory conditions. Here evidence is provided for the first time that the SixA phosphatase is a crucial regulatory factor that is involved in the ArcB signaling, particularly, under certain anaerobic respiratory growth conditions. We propose a novel mechanism, involving an HPt domain and a phospho‐histidine phosphatase, by which a given multi‐step His‐to‐Asp signaling can be modulated.

Staphylococcus aureus peptidoglycan stimulates granulocyte macrophage colony-stimulating factor production from human epidermal keratinocytes via mitogen-activated protein kinases.

Epidermal keratinocytes with atopic dermatitis (AD) overproduce mediators such as granulocyte macrophage colony‐stimulating factor (GM‐CSF), which are associated with pathology of AD. We found that peptidoglycan (PGN) of Staphylococcus aureus, which is frequently observed in lesion with AD, induced the production of numerous mediators such as GM‐CSF and regulated on activation, normal T‐cell expressed and secreted. Moreover, PGN phosphorylated extracellular‐signal‐regulated kinases and p38 mitogen‐activated protein kinase, which were involved in the induction of GM‐CSF expression. These results suggested that PGN of S. aureus directly exacerbates inflammation of inflammatory skin disease.

Histamine H1 Receptor-Stimulated Interleukin 8 and Granulocyte Macrophage Colony-Stimulating Factor Production by Bronchial Epithelial Cells Requires Extracellular Signal-Regulated Kinase Signaling via Protein Kinase C

Background: Histamine stimulates the release of several cytokines, such as interleukin (IL)-8 and granulocyte macrophage colony-stimulating factor, from bronchial epithelial cells. However, the functional individual histamine receptor subtype and intracellular signaling in bronchial epithelial cells are poorly defined. Methods: Using human primary epithelial cells and the NCI-H292 cell line, we examined the expression of histamine receptor subtypes and histamine-induced second messenger. We also evaluated the involvements of mitogen-activated protein kinase, protein kinase C (PKC) and epidermal growth factor receptor in cytokine expression caused by histamine. Results: Histamine H1 receptor (H1R) was the only subtype expressed in both types of cells. Histamine elevated intracellular calcium ion without affecting cAMP levels. Histamine induced the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Histamine also phosphorylated PKC and myristoylated alanine-rich C kinase substrate. Ro-31-8220, a PKC inhibitor, and PD98059, a mitogen-activated protein/ERK kinase inhibitor, suppressed the histamine-induced ERK activation and the production of granulocyte macrophage colony-stimulating factor and IL-8. On the contrary, histamine had no effect on the phosphorylation of epidermal growth factor receptor, and its specific inhibitor AG1478 failed to inhibit the histamine-induced ERK activation. Olopatadine, an H1 antagonist, completely blocked the histamine-related responses, whereas H2 and H3 antagonists did not. Histamine also augmented the IL-8 production caused by IL-4 or tumor necrosis factor-α. Conclusions: The H1R-PKC-ERK pathway may play crucial roles in eliciting cytokine production from bronchial epithelial cells stimulated by histamine, leading to airway inflammation.

Olopatadine Ameliorates Rat Experimental Cutaneous Inflammation by Improving Skin Barrier Function

Olopatadine hydrochloride (olopatadine) is an antiallergic agent with histamine H1 receptor antagonistic action. We investigated the possible efficacies of olopatadine on the chronic inflammatory dermatitis and the impaired skin barrier functions induced by repeated application of oxazolone in rats. Oxazolone-sensitized rats were challenged with oxazolone applied to the ear every 3 days. Olopatadine was orally administered once daily (1 and 3 mg/kg/day). The effects of the drug were quantified by measurements of ear thickness, levels of cytokines in the lesioned ear and the number of scratching episodes. As parameters of skin barrier function, transepidermal water loss (TEWL) and hyaluronic acid (HA) levels in the lesioned ear were measured. The effect of olopatadine on the production of HA by cultured dermal fibroblasts was also measured. Repeated topical application of oxazolone to rat ears induced local inflammation that was exemplified by swelling. In inflamed ears, the amount of IFNγ increased at both the protein and mRNA level, but IL-4 levels changed minimally. Olopatadine significantly decreased ear swelling and the number of scratching episodes. The drug also significantly inhibited the increase of IFNγ and nerve growth factor production in inflamed ears. Olopatadine significantly inhibited the increase in TEWL and the decrease in HA in lesioned ears. Furthermore, the drug stimulated the production of HA by cultured dermal fibroblasts. These results suggest that olopatadine suppressed inflammation and scratching not only by inhibiting cytokine production, but also by repairing skin barrier function.