Carola E. Matus

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family

During neutrophil activation and degranulation, MMP‐9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G‐protein‐coupled B1R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP‐9 and MPO by the B1R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin‐treated and ‐untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP‐9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP‐9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B1R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B1R. Our results demonstrate that in human neutrophils, activation of kinin B1R by LDBK initiates separate signaling cascades that trigger the release of MMP‐9 and MPO from tertiary and primary granules, respectively, suggesting that the B1R plays a pivotal role in inflammatory disorders.

Stimulation of the bradykinin B1 receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B1 (B1R) and B2 receptors. Expression of the B1R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B1R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B1R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B1R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B1R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B1R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B1R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B1R may be a valuable alternative for the treatment of breast cancer.

Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis.

Expression of HER2 and bradykinin B1 receptors in precursor lesions of gallbladder carcinoma

AIM To determine the expression of HER2 and bradykinin B(1) receptors (B(1)R) in the two pathogenic models of gallbladder cancer: the metaplasia-dysplasia-carcinoma and the adenoma-carcinoma pathways. METHODS Receptor proteins were visualized by immunohistochemistry on 5-μm sections of paraffin-embedded tissue. Expression of both receptors was studied in biopsy samples from 92 patients (6 males and 86 females; age ranging from 28 to 86 years, mean 56 years). High HER2 expression in specimens was additionally investigated by fluorescence in situ hybridization. Cell proliferation in each sample was assessed by using the Ki-67 proliferation marker. RESULTS HER2 receptor protein was absent in adenomas and in normal gallbladder epithelium. On the contrary, there was intense staining for HER2 on the basolateral membrane of epithelial cells of intestinal metaplasia (22/24; 91.7%) and carcinoma in situ (9/10; 90%), the lesions that displayed a significantly high proliferation index. Protein up-regulation of HER2 in the epithelium with metaplasia or carcinoma in situ was not accompanied by HER2 gene amplification. A similar result was observed in invasive carcinomas (0/12). The B(1)R distribution pattern mirrored that of HER2 except that B(1)R was additionally observed in the adenomas. The B(1)R appeared either as cytoplasmic dots or labeling on the apical cell membrane of the cells composing the epithelia with intestinal metaplasia (24/24; 100%) and carcinoma in situ (10/10; 100%) and in the epithelial cells of adenomas. In contrast, both HER2 (4/12; 33%) and B(1)R (1/12; 8.3%) showed a low expression in invasive gallbladder carcinomas. CONCLUSION The up-regulation of HER2 and B(1)R in precursor lesions of gallbladder carcinoma suggests cross-talk between these two receptors that may be of importance in the modulation of cell proliferation in gallbladder carcinogenesis.

Expression and bioregulation of the kallikrein-related peptidases family in the human neutrophil

The family of kallikrein-related peptidases (KLKs) has been identified in a variety of immunolabeled human tissue sections, but no previous study has experimentally confirmed their presence in the human neutrophil. We have investigated the expression and bioregulation of particular KLKs in the human neutrophil and, in addition, examined whether stimulation by a kinin B1 receptor (B1R) agonist or fMet-Leu-Phe (fMLP) induces their secretion. Western blot analysis of neutrophil homogenates indicated that the MM of the KLKs ranged from 27 to 50 kDa. RT-PCR showed that blood neutrophils expressed only KLK1, KLK4, KLK10, KLK13, KLK14 and KLK15 mRNAs, whereas the non-differentiated HL-60 cells expressed most of them, with exception of KLK3 and KLK7. Nevertheless, mRNAs for KLK2, KLK5, KLK6 and KLK9 that were previously undetectable appeared after challenging with a mixture of cytokines. Both kinin B1R agonist and fMLP induced secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the culture medium in similar amounts, whereas the B1R agonist caused the release of lower amounts of KLK2, KLK4 and KLK5. When secreted, the differing proteolytic activity of KLKs provides the human neutrophil with a multifunctional enzymatic capacity supporting a new dimension for its role in human disorders of diverse etiology.

Kinin B1 receptor regulates interactions between neutrophils and endothelial cells by modulating the levels of Mac-1, LFA-1 and intercellular adhesion molecule-1

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg9]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation.

Intracellular signaling pathways involved in the release of IL-4 and VEGF from human keratinocytes by activation of kinin B1 receptor: functional relevance to angiogenesis

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg9]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2′-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.

Activation of the human keratinocyte B1 bradykinin receptor induces expression and secretion of metalloproteases 2 and 9 by transactivation of epidermal growth factor receptor.

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch‐wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen‐activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up‐regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing.

Signaling Pathways Coupled to Activation of the Kinin B1 Receptor

Kinins are a group of bioactive peptides, which are formed by an endogenous enzymatic cascade consisting of precursor substrates called kininogens and the proteolytic kallikrein enzymes (kininogenases). So far, two kinin-releasing enzymes (kininogenases) have been characterized, plasma (hKB1/LKLB1) and tissue (hK1/KLK1) kallikreins (Fig. 1). Both enzymes are serine proteases that are found in glandular cells, neutrophils, and biological fluids. Kininogens are multifunctional proteins involved in cascade reactions during inflammation (Bhoola et al., 1992), and more recently in carcinogenesis (Bhoola et al., 1992; 2001; Leeb-Lundberg et al., 2005). Kinins namely bradykinin (BK) and kallidin (Lys-BK) are among the most potent pro-inflammatory vasoactive peptides generated during tissue injury and noxious stimulation. BK and Lys-BK undergo metabolic degradation at a variable rate by amino-, carboxyand endopeptidases found in tissues and biological fluids. The most physiologically relevant enzymes are carboxypeptidase N (from plasma) and carboxypeptidase M (from cell membranes), which remove the carboxy-terminal Arg present at end of the kinin molecule, resulting in the formation of the active metabolites desArg9-BK (DBK) and des-Arg10-kallidin, known also as Lys-des-Arg9-BK (LDBK) (Fig. 1). Furthermore, neutral endopeptidase 24.11 (CD10, enkephalinase) and angiotensin I converting enzyme, act as dipeptidyl carboxypeptidases by removing the dipeptide Phe8Arg9 from the carboxy terminus end of BK or Lys-BK (Couture et al., 2004). It is important to mention that endopeptidase 24.15 and angiotensin I converting enzyme cleave the dipeptide Ser6-Pro7 from bradykinin 1-7 (BK1-7) to produce bradykinin 1-5 (BK1-5), one of the final metabolites of BK and DBK that possesses the longer half-life of this peptide family. The half-life of kinins depends on the rate and site of destruction, most rapid in the circulation, but less so in the extracellular fluid space and by cells. Actually, in plasma the half-life of BK and LDBK is short (15 to 20 sec) whereas BK1-5, considered in the past an inactive fragment, is considered to have a half-life of 86 to 101 min (Shima et al., 1992; Murphey et al., 2000; 2006; Morinelli et al., 2002).

Functional interrelationships between the kallikrein-related peptidases family and the classical kinin system in the human neutrophil

Abstract In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.