Activation of kinin B receptor triggers differentiation of cultured human keratinocytes.

BACKGROUND: Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B(1) receptor (B(1)R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology. OBJECTIVES: To determine the consequence of kinin B(1)R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation. METHODS: Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin. RESULTS: The major proteins phosphorylated, after B(1)R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170- and 125-kDa proteins were identified as EGFR and p125(FAK), respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12-myristate 13-acetate, indicating the participation of protein kinase C. B(1)R stimulation did not increase [Ca(2+)]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr(845), Tyr(992) and Tyr(1068) of EGFR. B(1)R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B(1)R stimulation, produced an additional increase in filaggrin expression. CONCLUSIONS: The kinin B(1)R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.