A Novel Signaling Pathway: FIBROBLAST NICOTINIC RECEPTOR ��1 BINDS UROKINASE AND PROMOTES RENAL FIBROSIS*

The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRα1 during fibrogenesis. Silencing of the nAChRα1 gene led to significantly fewer interstitial αSMA⁺ myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRα1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRα1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. ³⁵S-Labeled uPA bound to nAChRα1 with a K_d of 1.6 × 10⁻⁸ m, which was displaced by the specific nAChRα1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [³H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRα1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRα1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease.Urokinase was first isolated from human urine in 1955 and identified as an activator of plasminogen (urokinase plasminogen activator (uPA)²) (1). This serine protease is abundantly produced by kidney tubular cells and secreted across the apical membrane into the urinary space. Other cellular sources include monocytes/macrophages, fibroblasts, and myofibroblasts (2). Despite high uPA levels, its primary physiological function in the kidney remains unknown. Suggested roles have been an inhibitor of kidney stone formation and urinary tract infections due to its proteolytic activity and endogenous antibiotic function, respectively (3, 4). Increased uPA activity has been reported in several pathological conditions, such as chronic kidney disease (2), atherosclerosis, and malignant tumors (5, 6). Endogenous plasma uPA levels may be elevated 2–4-fold in patients with chronic kidney disease due to increased uPA released from damaged kidneys (7, 8).Since its identification as a mediator of fibrin/fibrinogen degradation, uPA has been used in clinical settings as a fibrinolytic agent. The classical cellular urokinase receptor (uPAR) was first discovered on the surface of monocytes in 1985. Since then, a diverse array of biological functions triggered by uPA-uPAR interactions has been elucidated and shown to have important effects on cellular behavior during embryogenesis, angiogenesis, wound healing, and metastases (9–11). The specific role of uPA in fibrotic disorders appears to be organ-specific. uPA deficiency worsened bleomycin-induced lung fibrosis and reduced fibrosis in hearts damaged by viral myocarditis or left ventricular pressure overload, whereas there was no net effect on the severity of renal fibrosis induced by ureteral obstruction (UUO), although uPAR deficiency worsened fibrosis in that model (2). Despite its association with a broad repertoire of activities, the uPA mechanism of action remains incompletely understood. In particular, there is accumulating evidence that uPA may have protease- and uPAR-independent cellular effects. For example, macrophage uPA overexpression causes cardiac inflammation and fibrosis. Of particular note, this effect is independent of the classic uPA receptor uPAR and can be abrogated using a calcium channel blocker (12).Recent evidence suggests that additional uPA receptor(s) may exist (13–15). We reported that urokinase initiates renal fibroblast signaling via the MAPK/ERK pathway (16). This response appears to be mediated, at least in part, by an alternative urokinase receptor, since uPA can initiate mitogenesis in uPAR^−/− fibroblasts. Using phage display technology, Liang et al. (17) reported putative uPA-binding consensus sequences in 12 transmembrane receptors and suggested them as candidate alternative uPA receptor(s). Of these candidate receptors, several are already known as uPAR co-receptors: low density lipoprotein receptor-related protein, gp130, integrin αv, uPAR-associated protein (also known as Endo180 and Mrc2), and the insulin-like growth factor II/mannose 6-phosphate receptor. It is also possible that different uPA domains might simultaneously bind to uPAR and one of its co-receptors (18). The muscle type nicotinic receptor α1 (nAChRα1) was among the receptor candidates. The muscle type nAChR is a ligand-gated ion channel known to mediate signal transduction at the neuromuscular junction (19). This receptor is a pentametric glycoprotein comprising five membrane-spanning subunits (two α1, β1, γ, and δ) that form a ligand-gated ion channel. The currently known nAChRα1 ligands are nicotine and acetylcholine. The ligand-binding domain (interface of α1/γ or α1/δ) involves the two α1 chains, which form a specialized pocket of aromatic and hydrophobic residues structurally similar to uPAR (20, 21). Upon ligation, the receptor changes its conformation and becomes permeable to sodium and calcium ions. Receptor function is regulated by tyrosine phosphorylation and dephosphorylation by kinases and phosphatases, respectively (22). The nAChRα1 is expressed and activated during muscle differentiation during embryonic development and following mature muscle denervation (23). Vascular endothelium, macrophages, and fibroblasts are also known to express certain nAChR subtypes (24). We observed that nAChRα1 expression was significantly higher in the kidneys of uPAR-deficient mice that develop worse scarring during UUO (supplemental Fig. S1a).Evidence that nAChRα1 might function as an alternative uPA receptor was suggested by our microarray data that compared uPAR^−/− and uPAR^+/+ renal fibroblasts. The nAChRα1 was the only one of the 12 receptor candidates identified by Liang et al. (17) that was significantly up-regulated on the uPAR^−/− fibroblasts (n = 5, p < 0.01 by analysis of variance (ANOVA), >2-fold change) (supplemental Fig. S1b). Based on the assumption that the receptor may be up-regulated in damaged kidneys in the absence of uPAR and contribute to the development of more severe fibrosis, this study was designed to determine if a ligand-receptor relationship exists between uPA and nAChRα1 and to investigate its functional role in fibroblast growth and renal fibrosis. In vivo functional knockdown of the nAChRα1 was shown to significantly attenuate fibrosis after ureteral obstruction, an effect that was uPA-dependent. In vitro studies provided additional evidence that nAChRα1 was an uPA signaling receptor for fibroblasts, activating a complex of signaling proteins by tyrosine phosphorylation and calcium influx to stimulate proliferation and enhance contractility.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Biochemical Characterization and Function of Complexes Formed by Hyaluronan and the Heavy Chains of Inter-��-inhibitor (HC��HA) Purified from Extracts of Human Amniotic Membrane

Clinically, amniotic membrane (AM) suppresses inflammation, scarring, and angiogenesis. AM contains abundant hyaluronan (HA) but its function in exerting these therapeutic actions remains unclear. Herein, AM was extracted sequentially with buffers A, B, and C, or separately by phosphate-buffered saline (PBS) alone. Agarose gel electrophoresis showed that high molecular weight (HMW) HA (an average of ∼3000 kDa) was predominantly extracted in isotonic Extract A (70.1 ± 6.0%) and PBS (37.7 ± 3.2%). Western blot analysis of these extracts with hyaluronidase digestion or NaOH treatment revealed that HMW HA was covalently linked with the heavy chains (HCs) of inter-α-inhibitor (IαI) via a NaOH-sensitive bond, likely transferred by the tumor necrosis factor-α stimulated gene-6 protein (TSG-6). This HC·HA complex (nHC·HA) could be purified from Extract PBS by two rounds of CsCl/guanidine HCl ultracentrifugation as well as in vitro reconstituted (rcHC·HA) by mixing HMW HA, serum IαI, and recombinant TSG-6. Consistent with previous reports, Extract PBS suppressed transforming growth factor-β1 promoter activation in corneal fibroblasts and induced mac ro phage apo pto sis. However, these effects were abolished by hyaluronidase digestion or heat treatment. More importantly, the effects were retained in the nHC·HA or rcHC·HA. These data collectively suggest that the HC·HA complex is the active component in AM responsible in part for clinically observed anti-inflammatory and anti-scarring actions.Hyaluronan (HA)⁴ is widely distributed in extracellular matrices, tissues, body fluids, and even in intracellular compartments (reviewed in Refs. 1 and 2). The molecular weight of HA ranges from 200 to 10,000 kDa depending on the source (3), but can also exist as smaller fragments and oligosaccharides under certain physiological or pathological conditions (1). Investigations over the last 15 years have suggested that low M_r HA can induce the gene expression of proinflammatory mediators and proangiogenesis, whereas high molecular weight (HMW) HA inhibits these processes (4–7).Several proteins have been shown to bind to HA (8) such as aggrecan (9), cartilage link protein (10), versican (11), CD44 (12, 13), inter-α-inhibitor (IαI) (14, 15), and tumor necrosis factor-α stimulated gene-6 protein (TSG-6) (16, 17). IαI consists of two heavy chains (HCs) (HC1 and HC2), both of which are linked through ester bonds to a chondroitin sulfate chain that is attached to the light chain, i.e. bikunin. Among all HA-binding proteins, only the HCs of IαI have been clearly demonstrated to be covalently coupled to HA (14, 18). However, TSG-6 has also been reported to form stable, possibly covalent, complexes with HA, either alone (19, 20) or when associated with HC (21).The formation of covalent bonds between HCs and HA is mediated by TSG-6 (22–24) where its expression is often induced by inflammatory mediators such as tumor necrosis factor-α and interleukin-1 (25, 26). TSG-6 is also expressed in inflammatory-like processes, such as ovulation (21, 27, 28) and cervical ripening (29). TSG-6 interacts with both HA (17) and IαI (21, 24, 30–33), and is essential for covalently transferring HCs on to HA (22–24). The TSG-6-mediated formation of the HC·HA complex has been demonstrated to play a crucial role in female fertility in mice. The HC·HA complex is an integral part of an expanded extracellular “cumulus” matrix around the oocyte, which plays a critical role in successful ovulation and fertilization in vivo (22, 34). HC·HA complexes have also been found at sites of inflammation (35–38) where its pro- or anti-inflammatory role remain arguable (39, 40).Immunostaining reveals abundant HA in the avascular stromal matrix of the AM (41, 42).⁵ In ophthalmology, cryopreserved AM has been widely used as a surgical graft for ocular surface reconstruction and exerts clinically observable actions to promote epithelial wound healing and to suppress inflammation, scarring, and angiogenesis (for reviews see Refs. 43–45). However, it is not clear whether HA in AM forms HC·HA complex, and if so whether such an HC·HA complex exerts any of the above therapeutic actions. To address these questions, we extracted AM with buffers of increasing salt concentration. Because HMW HA was found to form the HC·HA complex and was mainly extractable by isotonic solutions, we further purified it from the isotonic AM extract and reconstituted it in vitro from three defined components, i.e. HMW HA, serum IαI, and recombinant TSG-6. Our results showed that the HC·HA complex is an active component in AM responsible for the suppression of TGF-β1 promoter activity, linkable to the scarring process noted before by AM (46–48) and by the AM soluble extract (49), as well as for the promotion of macrophage death, linkable to the inflammatory process noted by AM (50) and the AM soluble extract (51).     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.