Secreted Versus Membrane-anchored Collagenases: RELATIVE ROLES IN FIBROBLAST-DEPENDENT COLLAGENOLYSIS AND INVASION*

Fibroblasts degrade type I collagen, the major extracellular protein found in mammals, during events ranging from bulk tissue resorption to invasion through the three-dimensional extracellular matrix. Current evidence suggests that type I collagenolysis is mediated by secreted as well as membrane-anchored members of the matrix metalloproteinase (MMP) gene family. However, the roles played by these multiple and possibly redundant, degradative systems during fibroblast-mediated matrix remodeling is undefined. Herein, we use fibroblasts isolated from Mmp13^−/−, Mmp8^−/−, Mmp2^−/−, Mmp9^−/−, Mmp14^−/− and Mmp16^−/− mice to define the functional roles for secreted and membrane-anchored collagenases during collagen-resorptive versus collagen-invasive events. In the presence of a functional plasminogen activator-plasminogen axis, secreted collagenases arm cells with a redundant collagenolytic potential that allows fibroblasts harboring single deficiencies for either MMP-13, MMP-8, MMP-2, or MMP-9 to continue to degrade collagen comparably to wild-type fibroblasts. Likewise, Mmp14^−/− or Mmp16^−/− fibroblasts retain near-normal collagenolytic activity in the presence of plasminogen via the mobilization of secreted collagenases, but only Mmp14 (MT1-MMP) plays a required role in the collagenolytic processes that support fibroblast invasive activity. Furthermore, by artificially tethering a secreted collagenase to the surface of Mmp14^−/− fibroblasts, we demonstrate that localized pericellular collagenolytic activity differentiates the collagen-invasive phenotype from bulk collagen degradation. Hence, whereas secreted collagenases arm fibroblasts with potent matrix-resorptive activity, only MT1-MMP confers the focal collagenolytic activity necessary for supporting the tissue-invasive phenotype.In the postnatal state, fibroblasts are normally embedded in a self-generated three-dimensional connective tissue matrix composed largely of type I collagen, the major extracellular protein found in mammals (1–3). Type I collagen not only acts as a structural scaffolding for the associated mesenchymal cell populations but also regulates gene expression and cell function through its interactions with collagen binding integrins and discoidin receptors (2, 4). Consistent with the central role that type I collagen plays in defining the structure and function of the extracellular matrix, the triple-helical molecule is resistant to almost all forms of proteolytic attack and can display a decades-long half-life in vivo (4–6). Nonetheless, fibroblasts actively remodel type I collagen during wound healing, inflammation, or neoplastic states (2, 7–13).To date type I collagenolytic activity is largely confined to a small subset of fewer than 10 proteases belonging to either the cysteine proteinase or matrix metalloproteinase (MMP)² gene families (4, 14–18). As all collagenases are synthesized as inactive zymogens, complex proteolytic cascades involving serine, cysteine, metallo, and aspartyl proteinases have also been linked to collagen turnover by virtue of their ability to mediate the processing of the pro-collagenases to their active forms (13, 15, 19). After activation, each collagenase can then cleave native collagen within its triple-helical domain, thus precipitating the unwinding or “melting” of the resulting collagen fragments at physiologic temperatures (4, 15). In turn, the denatured products (termed gelatin) are susceptible to further proteolysis by a broader class of “gelatinases” (4, 15). Collagen fragments are then either internalized after binding to specific receptors on the cell surface or degraded to smaller peptides with potent biological activity (20–24).Previous studies by our group as well as others have identified MMPs as the primary effectors of fibroblast-mediated collagenolysis (20, 25, 26). Interestingly, adult mouse fibroblasts express at least six MMPs that can potentially degrade type I collagen, raising the possibility of multiple compensatory networks that are designed to preserve collagenolytic activity (25). Four of these collagenases belong to the family of secreted MMPs, i.e. MMP-13, MMP-8, MMP-2, and MMP-9, whereas the other two enzymes are members of the membrane-type MMP subgroup, i.e. MMP-14 (MT1-MMP) and MMP-16 (MT3-MMP) (13, 27–29). From a functional perspective, the specific roles that can be assigned to secreted versus membrane-anchored collagenases remain undefined. As such, fibroblasts were isolated from either wild-type mice or mice harboring loss-of-function deletions in each of the major secreted and membrane-anchored collagenolytic genes, and the ability of the cells to degrade type I collagen was assessed. Herein, we demonstrate that fibroblasts mobilize either secreted or membrane-anchored MMPs to effectively degrade type I collagen in qualitatively and quantitatively distinct fashions. However, under conditions where fibroblasts use either secreted and membrane-anchored MMPs to exert quantitatively equivalent collagenolytic activity, only MT1-MMP plays a required role in supporting a collagen-invasive phenotype. These data establish a new paradigm wherein secreted collagenases are functionally limited to bulk collagenolytic processes, whereas MT1-MMP uniquely arms the fibroblast with a focalized degradative activity that mediates subjacent collagenolysis as well as invasion.     

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.     

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO₂⁻) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H₂O and D₂O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H₂O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D₂O than in H₂O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.Denitrification is an anaerobic respiration pathway found in bacteria, archaea, and fungi, in which ATP synthesis is coupled to the sequential reduction of nitrate (NO₃⁻) and nitrite (NO₂⁻) (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (1–3).³ The first committed step in this reaction cascade is the formation of gaseous NO by nitrite reductase (NiR), the key enzyme of this pathway. Two distinct classes of periplasmic NiR are found in denitrifying bacteria, one containing cd₁ hemes as prosthetic groups (4–6) and the other utilizing two copper centers to catalyze the one-electron reduction of nitrite (7). Copper-containing NiRs are divided into two main groups according to the color of their oxidized type 1 copper center (T1Cu), with shades ranging from blue to green (3, 7). NiR from Alcaligenes xylosoxidans subsp. xylosoxidans (NCIMB 11015, AxNiR), which is analyzed in this study, is a member of the blue CuNiR group. The blue and green subclasses show a high degree of sequence similarity (70%) (8) and have similar trimeric structures with each monomer (∼36.5 kDa in AxNiR) consisting of two greek key β-barrel cupredoxin-like motifs as well as one long and two short α-helical regions (7, 9).Each NiR monomer contains two copper-binding sites per catalytic unit. One is a T1Cu center, which receives electrons from a physiological redox partner protein and is buried 7 Å beneath the protein surface (10), and the other copper is a type 2 center (T2Cu), constituting the catalytically active substrate-binding site (11). The physiological electron donor for the blue NiRs are the small copper protein azurin (14 kDa) (7) and cytochrome c₅₅₁ (7, 12, 13). The T1Cu, which is responsible for the color of NiR, serves as the electron delivery center and is coordinated by two histidine residues as well as one cysteine and one methionine residue. The catalytic T2Cu, which like all T2Cu centers has very weak optical bands, is ligated to three His residues and an H₂O/OH⁻ ligand in the resting state. This H₂O/OH⁻ ligand is held in place by hydrogen bonds to the active site residues, Asp-92 (AxNiR numbering) and His-249, and gets displaced by the substrate during catalytic turnover (14). The T2Cu is located at the base of a 13–14-Å substrate access channel at the interface of two monomers with one of the three His residues being part of the adjacent subunit (15, 16). The two copper centers are connected by a 12.6-Å covalent bridge provided by the T1Cu-coordinating Cys and by one of the T2Cu His ligands (17, 18). This linkage has been suggested to constitute the electron transfer (ET) pathway from the T1Cu center to the catalytically active T2Cu center via 11 covalent bonds (19).Intramolecular ET from T1- to T2Cu has been extensively examined using pulse radiolysis studies (7, 19–24). In a variety of NiR species, ET could be measured, both in the presence and absence of substrate, with observed ET rate constants (k_ET(obs)) ranging from ∼150 to ∼2000 s⁻¹. According to the Marcus semi-classical ET theory (25), the redox potentials (E′₀, redox midpoint potential at pH 7.0) of the copper centers affect both the thermodynamic equilibrium and the ET kinetics. In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (E′₀ (T1Cu) ∼240 mV and E′₀ (T2Cu) ∼230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (≥150 s⁻¹) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H₂O/OH⁻ ligand and unable to bind nitrite (26, 27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7, 16, 25, 27–30). However, k_ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitrite-bound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16, 25, 30, 31). These rearrangements include subtle changes in the Cys-His bridge linking T1- and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref. 29). Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19, 27, 29, 32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14, 16, 19, 26, 27, 33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34). This finding, however, contradicts the pulse radiolysis results at room temperature (see above), and the apparent discrepancy between solution studies and x-ray crystallographic data collected at cryogenic temperature remains to be resolved.The one-electron reduction of nitrite to NO involves two protons according to the chemical net equation NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O, if the T2Cu is ligated by an H₂O molecule in the resting state rather than an OH⁻ ion. Although the exact enzymatic mechanism is still somewhat controversial (35, 36), one suggested reaction sequence is given in Scheme 1. The potential participation of active site residues in catalyzing the proton transfer (PT) steps has been investigated by studying the pH dependence of NiR under steady-state conditions as well as by pulse radiolysis. The trends obtained for k_cat and k_ET(obs), are similar with pH optima between 5.2 and 6, indicating the involvement of two amino acid residues (21, 22, 37). Asp-92 and His-249 have been proposed as acid-base catalysts (18, 21, 22, 28, 38), and the abrupt drop in rates at increasing pH may indicate that OH⁻ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35, 40, 41).⁴Open in a separate windowSCHEME 1.A potential reaction mechanism proposed for CuNiRs. Adapted from Ref. 36. Nitrite is shown to bind to the oxidized T2Cu as nitrous acid, thus involving the first protonation step. It coordinates to the oxidized T2Cu center in a bidentate fashion. Following inter-copper ET yielding a reduced T2Cu center, the initially deprotonated Asp-92 accepts a proton, which is subsequently transferred to the substrate. His-249 may be a potential source of this second proton. PT and ET reactions may be reversible and they may be concerted rather than sequential as suggested by the arrows. See text for further information.There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET. An extensive analysis of the solvent kinetic isotope effect (SKIE) has been employed as a means of determining whether solvent-exchangeable protons and/or water molecules play a rate-limiting role in the catalytic turnover and/or in inter-copper ET.     

T-type Ca2+ Channels Promote Oxygenation-induced Closure of the Rat Ductus Arteriosus Not Only by Vasoconstriction but Also by Neointima Formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca²⁺ influx through several membrane Ca²⁺ channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca²⁺ channel (VDCC) in DA closure remains unclear. Here we found that the expression of α1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that α1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by α1G overexpression. Moreover, it was decreased by adding α1G-specific small interfering RNAs or using R(−)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca²⁺ concentration of DA SMCs was significantly decreased by adding α1G-specific siRNAs or using R(−)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(−)-efonidipine (10⁻⁶ m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(−)-efonidipine (10⁻⁶ m) significantly attenuated oxygenation-induced vasoconstriction by ∼27% using a vascular ring of fetal DA at term. Finally, R(−)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially α1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.The ductus arteriosus (DA)² is an essential vascular shunt between the aortic arch and the pulmonary trunk during a fetal period (1). After birth, the DA closes immediately in accordance with its smooth muscle contraction and vascular remodeling, whereas the connecting vessels such as the aorta and pulmonary arteries remain open. When the DA fails to close after birth, the condition is known as patent DA, which is a common form of congenital heart defect. Patent DA is also a frequent problem with significant morbidity and mortality in premature infants. Investigating the molecular mechanism of DA closure is important not only for vascular biology but also for clinical problems in pediatrics.Voltage-dependent Ca²⁺ channels (VDCCs) consist of multiple subtypes, named L-, N-, P/Q-, R-, and T-type. L-type VDCCs are known to play a primary role in regulating Ca²⁺ influx and thus vascular tone in the development of arterial smooth muscle including the DA (2–4). Our previous study demonstrated that all T-type VDCCs were expressed in the rat DA (5). α1G subunit, especially, was the most dominant isoform among T-type VDCCs. The abundant expression of α1G subunit suggests that it plays a role in the vasoconstriction and vascular remodeling of the DA. In this regard, Nakanishi et al. (6) demonstrated that 0.5 mm nickel, which blocks T-type VDCC, inhibited oxygen-induced vasoconstriction of the rabbit DA. On the other hand, Tristani-Firouzi et al. (7) demonstrated that T-type VDCCs exhibited little effect on oxygen-sensitive vasoconstriction of the rabbit DA. Thus, the role of T-type VDCCs in DA vasoconstriction has remained controversial.In addition to their role in determining the contractile state, a growing body of evidence has demonstrated that T-type VDCCs play an important role in regulating differentiation (8, 9), proliferation (10–12), migration (13, 14), and gene expression (15) in vascular smooth muscle cells (SMCs). Hollenbeck et al. (16) and Patel et al. (17) demonstrated that nickel inhibited platelet-derived growth factor-BB-induced SMC migration. Rodman et al. (18) demonstrated that α1G promoted SMC proliferation in the pulmonary artery. The DA dramatically changes its morphology during development. Intimal cushion formation, a characteristic feature of vascular remodeling of the DA (19–21), involves many cellular processes: an increase in SMC migration and proliferation, production of hyaluronic acid under the endothelial layer, impaired elastin fiber assembly, and so on (1, 19, 21–23). Although our previous study demonstrated that T-type VDCCs are involved in smooth muscle cell proliferation in the DA (5), the role of T-type VDCCs in vascular remodeling of the DA has remained poorly understood.In the present study, we hypothesized that T-type VDCCs, especially α1G subunit, associate with vascular remodeling and vasoconstriction in the DA. To test our hypothesis, we took full advantage of recent molecular and pharmacological developments. We chose the recently developed, highly selective T-type VDCC blocker R(−)-efonidipine instead of low dose nickel for our study. Selective inhibition or activation of α1G subunit was also obtained using small interfering RNA (siRNA) technology or by overexpression of the α1G subunit gene, respectively. We found that Ca²⁺ influx through T-type VDCCs promoted oxygenation-induced DA closure through SMC migration and vasoconstriction.