The family of kallikrein-related peptidases and kinin peptides as modulators of epidermal homeostasis.

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.

KCa3.1 differentially regulates trachea and bronchi epithelial gene expression in a chronic-asthma mouse model.

Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4, in regulating the gene expression of mouse airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild-type and Kcnn4-/- mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. In addition, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.

Kinin B1 Receptor Signaling in Skin Homeostasis and Wound Healing.

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment.

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

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.

Overview of tissue kallikrein and kallikrein-related peptidases in breast cancer.

The kallikrein family comprises tissue kallikrein and 14 kallikrein-related peptidases (KLKs) recognized as a subgroup of secreted trypsin- or chymotrypsin-like serine proteases. KLKs are expressed in many cellular types where they regulate important physiological activities such as semen liquefaction, immune response, neural development, blood pressure, skin desquamation and tooth enamel formation. Tissue kallikrein, the oldest member and kinin-releasing enzyme, and KLK3/PSA, a tumor biomarker for prostate cancer are the most prominent components of the family. Additionally, other KLKs have shown an abnormal expression in neoplasia, particularly in breast cancer. Thus, increased levels of some KLKs may increase extracellular matrix degradation, invasion and metastasis; other KLKs modulate cell growth, survival and angiogenesis. On the contrary, KLKs can also inhibit angiogenesis and produce tumor suppression. However, there is a lack of knowledge on how KLKs are regulated in tumor microenvironment by molecules present at the site, namely cytokines, inflammatory mediators and growth factors. Little is known about the signaling pathways that control expression/secretion of KLKs in breast cancer, and further how activation of PAR receptors may contribute to functional activity in neoplasia. A better understanding of these molecular events will allow us to consider KLKs as relevant therapeutic targets for breast cancer.

Possible role of phytoestrogens in breast cancer via GPER-1/GPR30 signaling.

Estrogens generated within endocrine organs and the reproductive system act as ligands for at least three types of estrogen receptors. Estrogen receptors α (ERα) and ß (ERß) belong to the so-called classical family of estrogen receptors, whereas the G protein-coupled receptor GPR30, also known as GPER-1, has been described as a novel estrogen receptor sited in the cell membrane of target cells. Furthermore, these receptors are under stimulation of a family of exogenous estrogens, known as phytoestrogens, which are a diverse group of non-steroidal plant compounds derived from plant food consumed by humans and animals. Because phytoestrogens are omnipresent in our daily diet, they are becoming increasingly important in both human health and disease. Recent evidence indicates that in addition to classical estrogen receptors, phytoestrogens also activate GPER-1 a relevant observation since GPER-1 is involved in several physiopathological disorders and especially in estrogen-dependent diseases such as breast cancer.The first estrogen receptors discovered were the classical ERα and ERß, but from an evolutionary point of view G protein-coupled receptors trace their origins in history to over a billion years ago suggesting that estrogen receptors like GPER-1 may have been the targets of choice for ancient phytoestrogens and/or estrogens.This review provides a comprehensive and systematic literature search on phytoestrogens and its relationship with classical estrogen receptors and GPER-1 including its role in breast cancer, an issue still under discussion.

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.

Pre-stimulation of the kallikrein system in cisplatin-induced acute renal injury: an approach to renoprotection.

Antineoplastic treatment with cisplatin is frequently complicated by nephrotoxicity. Although oxidative stress may be involved, the pathogenic mechanisms responsible for renal damage have not been completely clarified. In order to investigate the role of the renal kinin system in this condition, a group of rats was submitted to high potassium diet to stimulate the synthesis and excretion of tissue kallikrein 1 (rKLK1) previous to an intraperitoneal injection of 7 mg/kg cisplatin. A significant reduction in lipoperoxidation, evidenced by urinary excretion of malondialdehyde and renal immunostaining of hidroxy-nonenal, was accompanied by a decline in apoptosis. Coincident with these findings we observed a reduction in the expression of renal KIM-1 suggesting that renoprotection may be occurring. Stimulation or indemnity of the renal kinin system deserves to be evaluated as a complementary pharmacological measure to diminish cisplatin nephrotoxicity.

Antihypertensive and renoprotective effect of the kinin pathway activated by potassium in a model of salt sensitivity following overload proteinuria.

The albumin overload model induces proteinuria and tubulointersitial damage, followed by hypertension when rats are exposed to a hypersodic diet. To understand the effect of kinin system stimulation on salt-sensitive hypertension and to explore its potential renoprotective effects, the model was induced in Sprague-Dawley rats that had previously received a high-potassium diet to enhance activity of the kinin pathway, followed with/without administration of icatibant to block the kinin B2 receptor (B2R). A disease control group received albumin but not potassium or icatibant, and all groups were exposed to a hypersodic diet to induce salt-sensitive hypertension. Potassium treatment increased the synthesis and excretion of tissue kallikrein (Klk1/rKLK1) accompanied by a significant reduction in blood pressure and renal fibrosis and with downregulation of renal transforming growth factor-ß (TGF-ß) mRNA and protein compared with rats that did not receive potassium. Participation of the B2R was evidenced by the fact that all beneficial effects were lost in the presence of the B2R antagonist. In vitro experiments using the HK-2 proximal tubule cell line showed that treatment of tubular cells with 10 nM bradykinin reduced the epithelial-mesenchymal transdifferentiation and albumin-induced production of TGF-ß, and the effects produced by bradykinin were prevented by pretreatment with the B2R antagonist. These experiments support not only the pathogenic role of the kinin pathway in salt sensitivity but also sustain its role as a renoprotective, antifibrotic paracrine system that modulates renal levels of TGF-ß.

Vasopressin activates Akt/mTOR pathway in smooth muscle cells cultured in high glucose concentration.

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration.

Minireview: functional roles of tissue kallikrein, kinins, and kallikrein-related peptidases in lung cancer.

Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics.

The splice variant of the V2 vasopressin receptor adopts alternative topologies.

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.

The Impact of Estrogen and Estrogen-Like Molecules in Neurogenesis and Neurodegeneration: Beneficial or Harmful?

Estrogens and estrogen-like molecules can modify the biology of several cell types. Estrogen receptors alpha (ERα) and beta (ERß) belong to the so-called classical family of estrogen receptors, while the G protein-coupled estrogen receptor 1 (GPER-1) represents a non-classical estrogen receptor mainly located in the plasma membrane. As estrogen receptors are ubiquitously distributed, they can modulate cell proliferation, differentiation, and survival in several tissues and organs, including the central nervous system (CNS). Estrogens can exert neuroprotective roles by acting as anti-oxidants, promoting DNA repair, inducing the expression of growth factors, and modulating cerebral blood flow. Additionally, estrogen-dependent signaling pathways are involved in regulating the balance between proliferation and differentiation of neural stem/progenitor cells (NSPCs), thus influencing neurogenic processes. Since several estrogen-based therapies are used nowadays and estrogen-like molecules, including phytoestrogens and xenoestrogens, are omnipresent in our environment, estrogen-dependent changes in cell biology and tissue homeostasis have gained attention in human health and disease. This article provides a comprehensive literature review on the current knowledge of estrogen and estrogen-like molecules and their impact on cell survival and neurodegeneration, as well as their role in NSPCs proliferation/differentiation balance and neurogenesis.

Involvement of Kallikrein-Related Peptidases in Nervous System Disorders.

Kallikrein-related peptidases (KLKs) are a family of serine proteases that when dysregulated may contribute to neuroinflammation and neurodegeneration. In the present review article, we describe what is known about their physiological and pathological roles with an emphasis on KLK6 and KLK8, two KLKs that are highly expressed in the adult central nervous system (CNS). Altered expression and activity of KLK6 have been linked to brain physiology and the development of multiple sclerosis. On the other hand, altered levels of KLK6 in the brain and serum of people affected by Alzheimer's disease and Parkinson's disease have been documented, pointing out to its function in amyloid metabolism and development of synucleinopathies. People who have structural genetic variants of KLK8 can suffer mental illnesses such as intellectual and learning disabilities, seizures, and autism. Increased expression of KLK8 has also been implicated in schizophrenia, bipolar disorder, and depression. Also, we discuss the possible link that exists between KLKs activity and certain viral infections that can affect the nervous system. Although little is known about the exact mechanisms that mediate KLKs function and their participation in neuroinflammatory and neurodegenerative disorders will open a new field to develop novel therapies to modulate their levels and/or activity and their harmful effects on the CNS.

Continuous Exposure of Breast Cancer Cells to Tamoxifen Upregulates GPER-1 and Increases Cell Proliferation.

GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor α (ERα)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ERα-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ERα-positive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca2+]i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca2+]i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca2+]i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ERα expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ERα/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen.

Stimulation of the bradykinin B(1) 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 B(1) (B(1)R) and B(2) receptors. Expression of the B(1)R 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 B(1)R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B(1)R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B(1)R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B(1)R 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. B(1)R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B(1)R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B(1)R may be a valuable alternative for the treatment of breast cancer.

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways.

Activation of V(1a) receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. We found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V(1a) receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and beta-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways.

Functional expression of the free fatty acids receptor-1 and -4 (FFA1/GPR40 and FFA4/GPR120) in bovine endometrial cells.

Endometrial epithelial cells play a key defensive role as part of the innate immune response of cow uterus. An association between risk of acquiring infectious diseases and increased levels of free fatty acids postpartum has been suggested, and the use of omega-3 fatty acids such as docosahexaenoic acid (DHA) has been proposed as a beneficial strategy to improve immunity and fertility. The goal of our study was to demonstrate the presence of free fatty acid (FFA)-1 and 4 receptors in endometrial cells and to investigate their role on DHA interference in lipopolysaccharide (LPS)-induced inflammatory endometrial activation. We demonstrated that the bovine endometrial (BEND) cells line and bovine endometrium express both FFA1 and FFA4 receptors. FFA1 and FFA4 receptors were localized in the epithelium lining the endometrial cavity and in endometrial glands whereas in BEND cells a characteristic cell membrane localization of both receptors was observed. DHA, a FFA4 natural agonist, increased intracellular calcium mobilization in BEND cells, but the FFA1 agonists oleic and linoleic acids did not increase this response. DHA-induced intracellular calcium mobilization was inhibited by the FFA4 and FFA1 antagonists AH7614 and GW1100, respectively. DHA significantly reduced LPS-induced prostaglandin E2 (PGE2) production, but none of the antagonists reduced the effect produced by DHA. On the contrary, linoleic acid increased LPS-induced PGE2 production. In conclusion, endometrial cells express FFA4 and FFA1 receptors, and DHA induces intracellular calcium release via FFA4 and FFA1 receptors. DHA reduces PGE2, but this response was not mediated by FFA4 or FFA1 receptors.

Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney.

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.