Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2.

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis (Pg)-induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides (Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, ß-proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

Human Papillomavirus 16 E6 Induces FoxM1B in Oral Keratinocytes through GRHL2.

High-risk human papillomavirus (HPV) is a major risk factor for oral and pharyngeal cancers (OPCs), yet the detailed mechanisms by which HPV promotes OPCs are not understood. Forkhead box M1B (FoxM1B) is an oncogene essential for cell cycle progression and tumorigenesis, and it is aberrantly overexpressed in many tumors. We previously showed that FoxM1B was the putative target of an epithelial-specific transcription factor, Grainyhead-like 2 (GRHL2). In the current study, we demonstrate that HPV type 16 (HPV-16) E6 induces FoxM1B in human oral keratinocytes (HOKs) and tonsillar epithelial cells (TECs) in part through GRHL2. FoxM1B was barely detectable in cultured normal human oral keratinocytes (NHOKs) and progressively increased in immortalized HOKs harboring HPV-16 genome (HOK-16B) and tumorigenic HOK-16B/BaP-T cells. Retroviral expression of HPV-16 E6 and/or E7 in NHOKs, TECs, and hypopharyngeal carcinoma cells (FaDu) revealed induction of FoxM1B and GRHL2 by the E6 protein but not E7. Both GRHL2 and FoxM1B were strongly induced in the epidermis of HPV-16 E6 transgenic mice and HPV+ oral squamous cell carcinomas. Ectopic expression of FoxM1B led to acquisition of transformed phenotype in HOK-16B cells. Loss of FoxM1B by lentiviral short hairpin RNA vector or chemical inhibitor led to elimination of tumorigenic characteristics of HOK-16B/BaP-T cells. Luciferase reporter assay revealed that GRHL2 directly bound and regulated the FoxM1B gene promoter activity. Using epithelial-specific Grhl2 conditional knockout mice, we exposed wild-type (WT) and Grhl2 KO mice to 4-nitroquinolin 1-oxide (4-NQO), which led to induction of FoxM1B in the tongue tissues and rampant oral tumor development in the WT mice. However, 4-NQO exposure failed to induce tongue tumors or induction of FoxM1B expression in Grhl2 KO mice. Collectively, these results indicate that HPV-16 induces FoxM1B in part through GRHL2 transcriptional activity and that elevated FoxM1B level is required for oropharyngeal cancer development.

Biomarkers in acute kidney injury - pathophysiological basis and clinical performance.

Various biomarkers of acute kidney injury (AKI) have been discovered and characterized in the recent past. These molecules can be detected in urine or blood and signify structural damage to the kidney. Clinically, they are proposed as adjunct diagnostics to serum creatinine and urinary output to improve the early detection, differential diagnosis and prognostic assessment of AKI. The most obvious requirements for a biomarker include its reflection of the underlying pathophysiology of the disease. Hence, a biomarker of AKI should derive from the injured kidney and reflect a molecular process intimately connected with tissue injury. Here, we provide an overview of the basic pathophysiology, the cellular sources and the clinical performance of the most important currently proposed biomarkers of AKI: neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), liver-type fatty acid-binding protein (L-FABP), interleukin-18 (IL-18), insulin-like growth factor-binding protein 7 (IGFBP7), tissue inhibitor of metalloproteinase 2 (TIMP-2) and calprotectin (S100A8/9). We also acknowledge each biomarker's advantages and disadvantages as well as important knowledge gaps and perspectives for future studies.

Neutrophil gelatinase-associated lipocalin: pathophysiology and clinical applications.

Neutrophil gelatinase-associated lipocalin (NGAL), a 25 kDa protein produced by injured nephron epithelia, is one of the most promising new markers of renal epithelial injury. In contrast to serum creatinine and urinary output, which are the measures of kidney function, NGAL is specifically induced in the damaged nephron and then released into blood and urine, where it can be readily measured. Careful proof-of-concept studies using defined animal models have uncovered the sources and trafficking of NGAL in acute kidney injury (AKI) and have addressed the contributions of renal and non-renal sources. Clinical studies indicate that NGAL, unlike creatinine, is a marker responsive to tissue stress and nephron injury, but less so to adaptive hemodynamic responses. In certain clinical settings, NGAL is an earlier marker compared with serum creatinine. In addition, clinical studies have shown that NGAL is a powerful predictor of poor clinical outcomes, which can be used to risk stratify patients when combined with serum creatinine. NGAL has important limitations, including its responsiveness in systemic inflammation, which is partially uncoupled from its response to kidney injury and which needs to be considered when interpreting NGAL results clinically. This review covers the biology and pathophysiology of NGAL and summarizes the results of the growing body of clinical studies that have addressed the utility of NGAL in the early diagnosis of AKI, in the distinction of intrinsic AKI and in the prognostic assessment of broad patient populations.

Novel signalling mechanisms and targets in renal ischaemia and reperfusion injury.

Acute kidney injury (AKI) induced by ischaemia and reperfusion (I/R) injury is a common and severe clinical problem. Vascular dysfunction, immune system activation and tubular epithelial cell injury contribute to functional and structural deterioration. The search for novel therapeutic interventions for I/R-induced AKI is a dynamic area of experimental research. Pharmacological targeting of injury mediators and corresponding intracellular signalling in endothelial cells, inflammatory cells and the injured tubular epithelium could provide new opportunities yet may also pose great translational challenge. Here, we focus on signalling mediators, their receptors and intracellular signalling pathways which bear potential to abrogate cellular processes involved in the pathogenesis of I/R-induced AKI. Sphingosine 1 phosphate (S1P) and its respective receptors, cytochrome P450 (CYP450)-dependent vasoactive eicosanoids, NF-κB- and protein kinase-C (PKC)-related pathways are representatives of such 'druggable' pleiotropic targets. For example, pharmacological agents targeting S1P and PKC isoforms are already in clinical use for treatment for autoimmune diseases and were previously subject of clinical trials in kidney transplantation where I/R-induced AKI occurs as a common complication. We summarize recent in vitro and in vivo experimental studies using pharmacological and genomic targeting and highlight some of the challenges to clinical application of these advances.

Unraveling the role of connective tissue growth factor in diabetic nephropathy.

Marked upregulation of the secreted extracellular matrix-associated protein connective tissue growth factor (CTGF) in the glomerulus coincides with the onset of diabetic nephropathy. Recent studies, including the one by Yokoi et al., shed light on the role of CTGF in glomerular injury.

The multiple actions of angiotensin II in atherosclerosis.

Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system, has been implied in the pathogenesis of atherosclerosis on various levels. There is abundant experimental evidence that pharmacological antagonism of Ang II formation by angiotensin converting enzyme inhibition or blockade of the cellular effects of Ang II by angiotensin type 1 receptor blockade inhibits formation and progression of atherosclerotic lesions. Angiotensin promotes generation of oxidative stress in the vasculature, which appears to be a key mediator of Ang II-induced endothelial dysfunction, endothelial cell apoptosis, and lipoprotein peroxidation. Ang II also induces cellular adhesion molecules, chemotactic and proinflammatory cytokines, all of which participate in the induction of an inflammatory response in the vessel wall. In addition, Ang II triggers responses in vascular smooth muscle cells that lead to proliferation, migration, and a phenotypic modulation resulting in production of growth factors and extracellular matrix. While all of these effects contribute to neointima formation and development of atherosclerotic lesions, Ang II may also be involved in acute complications of atherosclerosis by promoting plaque rupture and a hyperthrombotic state. Accordingly, Ang II appears to have a central role in the pathophysiology of atherosclerosis.

The Discovery of Renin 100 Years Ago.

In 1898, Tigerstedt and Bergman published their observation that kidney extracts produce pressor effects. They characterized the substance and named it "renin." Although this was the beginning of understanding the role of the kidney in hypertension and the renin-angiotensin system, their discovery lay dormant for nearly 40 years.

Single-cell characterization of endothelin system gene expression in the cerebellum in situ.

To evaluate the expression of components of the endothelin (ET) system in single Purkinje neurons and Bergmann glial cells in situ, patch-clamp recording was combined with a multiplex RT-PCR approach. Cerebellar slices were rapidly isolated from 20- to 28-day-old mice. Cells were characterized morphologically and electrophysiologically and cell contents were aspirated and immediately reverse-transcribed. The cDNA was used as a template in a multiplex PCR reaction containing primers specific for ET-1, ET-2, and ET-3, ET-converting enzyme 1 (ECE-1) and ECE-2, and ETA and ETB receptors. The resulting PCR products were used as templates in a second PCR reaction containing only one pair of nested primers. Specific single bands were obtained from positive cells, which was confirmed by DNA sequencing of the PCR products. Of the 25 Purkinje neurons assayed, 84% were positive for ECE-1 mRNA and 68% for ECE-2 mRNA. No ET and ETA receptor mRNAs were detected, and only one cell was positive for ETB receptor mRNA. In Bergmann glial cells, ETB receptor mRNA was predominant. A total of 68% of the 25 cells assayed were positive. Sixteen percent were positive for ETA receptor mRNA, 8% for ECE-1 mRNA, and 12% for ECE-2 mRNA. Again, no ET mRNAs were detected. These results confirm the role of the ETB receptor in Bergmann glial cells and provide evidence for expression of ECE-1 and ECE-2 in Purkinje neurons.

Transcriptional regulation of endothelin-1 by erythropoietin in endothelial cells.

Recombinant human erythropoietin (rHuEpo) has been widely used in patients undergoing chronic hemodialysis treatment to correct anemia. In a subgroup of patients, i.v. administration of rHuEpo leads to manifestation or worsening of hypertension. The underlying mechanism of this remains unclear but it has been suggested that it is associated with increased expression of the vasoconstrictor endothelin (ET) in endothelial cells (ECs). There is also evidence for expression of specific rHuEpo receptors on ECs. The aim of this work was to study the time course and mechanisms of ET-1 regulation on the mRNA level in bovine aortic endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) stimulated with pharmacologic doses of rHuEpo (1-10 IU/ml). Compared to vehicle-treated controls, rHuEpo-treatment of ECs increases preproET-1 mRNA expression up to 170%, as shown by Northern blotting. To study the transcriptional regulation of ET-1 expression by rHuEpo, ECs were transfected with a luciferase construct driven by the rat ET-1 promoter and subsequently stimulated with rHuEpo. Compared to controls, luciferase activity increased up to 200% (n = 6; p < 0.05), suggesting transcriptional regulation of preproET-1 mRNA-expression by rHuEpo. Our data support the hypothesis that ET contributes to the hypertensive side effects of rHuEpo treatment and that this interaction occurs at the transcriptional level.