Immune response to intrapharyngeal LPS in neonatal and juvenile mice.

Neonates and infants have a higher morbidity and mortality associated with lower respiratory tract illnesses compared with older children. To identify age-related and longitudinal differences in the cellular immune response to acute lung injury (ALI), neonatal and juvenile mice were given Escherichia coli LPS using a novel, minimally invasive aspiration technique. Neonatal and juvenile mice received between 3.75 and 7.5 mg/kg LPS by intrapharyngeal aspiration. Airway and lung cells were isolated and characterized by flow cytometry, cytokine/chemokine mRNA expression from lung homogenates was quantified, and lung morphometry and injury scores were performed. LPS-treated neonatal mice underwent adoptive transfer with adult T regulatory cells (Tregs). After LPS aspiration, lung monocytes isolated from neonatal mice had a predominant M2 phenotype, whereas lung monocytes from juvenile mice displayed a mixed M1/M2 phenotype. At 72 hours after LPS exposure, neonatal lungs were slower to resolve inflammation and expressed lower mRNA levels of CCL2, CCL5, CXCL10, and IL-10. Juvenile, but not neonatal, mice demonstrated a significant increase in airway Tregs after LPS exposure. Adoptive transfer of adult Tregs into LPS-challenged neonatal mice resulted in reduced lung inflammation and improved weight gain. These findings underscore several vulnerabilities in the neonatal immune response to LPS-induced ALI. Most striking was the deficiency in airway Tregs after LPS aspiration. Adoptive transfer of adult Tregs mitigated LPS-induced ALI in neonatal mice, highlighting the importance of age-related differences in Tregs and their response to ALI during early postnatal development.

Extracellular matrix lumican promotes bacterial phagocytosis, and Lum-/- mice show increased Pseudomonas aeruginosa lung infection severity.

Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum(-/-)) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum(-/-) peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum(+/+) MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum(-/-) MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K(A) = 2.15 × 10(6) M(-1)), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum(-/-) MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.

Dual activation of CFTR and CLCN2 by lubiprostone in murine nasal epithelia.

Multiple sodium and chloride channels on the apical surface of nasal epithelial cells contribute to periciliary fluid homeostasis, a function that is disrupted in patients with cystic fibrosis (CF). Among these channels is the chloride channel CLCN2, which has been studied as a potential alternative chloride efflux pathway in the absence of CFTR. The object of the present study was to use the nasal potential difference test (NPD) to quantify CLCN2 function in an epithelial-directed TetOn CLCN2 transgenic mouse model (TGN-K18rtTA-hCLCN2) by using the putative CLCN2 pharmacological agonist lubiprostone and peptide inhibitor GaTx2. Lubiprostone significantly increased chloride transport in the CLCN2-overexpressing mice following activation of the transgene by doxycycline. This response to lubiprostone was significantly inhibited by GaTx2 after CLCN2 activation in TGN-CLCN2 mice. Cftr(-/-) and Clc2(-/-) mice showed hyperpolarization indicative of chloride efflux in response to lubiprostone, which was fully inhibited by GaTx2 and CFTR inhibitor 172 + GlyH-101, respectively. Our study reveals lubiprostone as a pharmacological activator of both CFTR and CLCN2. Overexpression and activation of CLCN2 leads to improved mouse NPD readings, suggesting it is available as an alternative pathway for epithelial chloride secretion in murine airways. The utilization of CLCN2 as an alternative chloride efflux channel could provide clinical benefit to patients with CF, especially if the pharmacological activator is administered as an aerosol.

Effects of detergents on catalytic activity of human endometase/matrilysin 2, a putative cancer biomarker.

Matrix metalloproteinases (MMPs) are a family of hydrolytic enzymes that play significant roles in development, morphogenesis, inflammation, and cancer invasion. Endometase (matrilysin 2 or MMP-26) is a putative early biomarker for human carcinomas. The effects of the ionic and nonionic detergents on catalytic activity of endometase were investigated. The hydrolytic activity of endometase was detergent concentration dependent, exhibiting a bell-shaped curve with its maximum activity near the critical micelle concentration (CMC) of nonionic detergents tested. The effect of Brij-35 on human gelatinase B (MMP-9), matrilysin (MMP-7), and membrane-type 1 MMP (MT1-MMP) was further explored. Their maximum catalysis was observed near the CMC of Brij-35 ( approximately 90muM). Their IC(50) values were above the CMC. The inhibition mechanism of MMP-7, MMP-9, and MT1-MMP by Brij-35 was a mixed type as determined by Dixon's plot; however, the inhibition mechanism of endometase was noncompetitive with a K(i) value of 240muM. The catalytic activities of MMPs are influenced by detergents. Monomer of detergents may activate and stabilize MMPs to enhance catalysis, but micelle of detergents may sequester enzyme and block the substrate binding site to impede catalysis. Under physiological conditions, a lipid or membrane microenvironment may regulate enzymatic activity.

Differential gene expression patterns of the developing and adult mouse cornea compared to the lens and tendon.

The cornea continues to mature after birth to develop a fully functional, refractive and protective barrier tissue. Here we investigated the complex biological events underlying this process by profiling global genome-wide gene expression patterns of the immature postnatal day 10 and 7-week old adult mouse cornea. The lens and tendon were included in the study to increase the specificity of genes identified as upregulated in the corneal samples. Notable similarities in gene expression between the cornea and the tendon were in the mesenchymal extracellular matrix collagen (types I, III, V, VI) and proteoglycan (lumican, decorin and biglycan) genes. Expression similarities in the cornea and lens were limited to certain epithelial genes and the crystallins. Approximately 76 genes were over expressed in the cornea samples that showed basal expression levels in the lens and tendon. Thirty-two of these were novel with no known functions in the cornea. These include genes with a potential role in protection against oxidative stress (Dhcr24, Cdo1, Akr1b7, Prdx6), inflammation (Ltb4dh, Wdr1), ion transport (Pdzk1ip1, Slc12a2, Slc25a17) and transcription (Zfp36l3, Pdzk1ip1). Direct comparison of the cornea of two ages showed selective upregulation of 50 and 12 genes in the P10 and adult cornea, respectively. Of the upregulated P10 genes several encode extracellular matrix collagens and proteoglycans that are stable components of the adult cornea and their high transcriptional activity at P10 indicate a period of actie corneal growth and matrix deposition in the young cornea. Much less is known about the genes selectively over expressed in the adult cornea; some relate to immune response and innervations (Npy), and possibly to electron transport (Cyp24a1, Cyp2f2) and others of yet unknown functions in the cornea (Rgs10, Psmb8, Xlr4). This study detected expression of genes with known functions in the cornea, providing additional validation of the microarray experiments. Importantly, it identified several novel genes whose functions have not been investigated in the cornea.

Calcium regulates tertiary structure and enzymatic activity of human endometase/matrilysin-2 and its role in promoting human breast cancer cell invasion.

Human MMP-26 (matrix metalloproteinase-26) (also known as endometase or matrilysin-2) is a putative biomarker for human carcinomas of breast, prostate and other cancers of epithelial origin. Calcium modulates protein structure and function and may act as a molecular signal or switch in cells. The relationship between MMPs and calcium has barely been studied and is absent for MMP-26. We have investigated the calcium-binding sites and the role of calcium in MMP-26. MMP-26 has one high-affinity and one low-affinity calcium binding site. High-affinity calcium binding was restored at physiologically low calcium conditions with a calcium-dissociation constant of 63 nM without inducing secondary and tertiary structural changes. High-affinity calcium binding protects MMP-26 against thermal denaturation. Mutants of this site (D165A or E191A) lose enzymatic activity. Low-affinity calcium binding was restored at relatively high calcium concentrations and showed a K(d2) (low-affinity calcium-dissociation constant) value of 120 microM, which was accompanied with the recovery of enzymatic activity reversibly and tertiary structural changes, but without secondary structural rearrangements. Mutations at the low-affinity calcium-binding site (C3 site), K189E or D114A, induced enhanced affinity for the Ca2+ ion or an irreversible loss of enzymatic activity triggered by low-affinity calcium binding respectively. Mutation at non-calcium-binding site (V184D at C2 site) showed that C2 is not a true calcium-binding site. Observations from homology-modelled mutant structures correlated with these experimental results. A human breast cancer cell line, MDA-MB-231, transfected with wild-type MMP-26 cDNA showed a calcium-dependent invasive potential when compared with controls that were transfected with an inactive form of MMP-26 (E209A). Calcium-independent high invasiveness was observed in the K189E mutant MDA-MB-231 cell line.

Digitoxin for Airway Inflammation in Cystic Fibrosis: Preliminary Assessment of Safety, Pharmacokinetics, and Dose Finding.

RATIONALE: Cystic fibrosis (CF) lung disease progresses by a combination of airway inflammation, bacterial colonization, and infection. Airway inflammation is predominantly neutrophilic and complicates airway clearance therapies through cellular debris; excessive DNA; excessive and viscous mucus; and high concentrations of neutrophils, IL-8, and related cytokines liberated along the nuclear factor-κB signaling pathway. OBJECTIVES: We conducted a preliminary, single-site, randomized, double-blind, placebo-controlled study to evaluate the effects over 28 days of two dose levels (0.05 mg and 0.1 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on safety, pharmacokinetics, and inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease. METHODS: Patients with CF 18-45 years old with any genotype combination were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure (1) the pharmacokinetics of digitoxin in sera of patients with stable CF; (2) safety indices, including ECG changes and sputum microbiology; (3) the effect of digitoxin on gene expression in nasal epithelial cells of patients with stable CF; and (4) quality-of-life scores using the Cystic Fibrosis Questionnaire-Revised. MEASUREMENTS AND MAIN RESULTS: It took several weeks to achieve a therapeutic serum level of digitoxin in subjects with CF. No safety concerns emerged during the study. Digitoxin treatment showed a trend toward reduction in sputum free neutrophil elastase and neutrophil counts, but not a reduction in sputum IL-8. Digitoxin treatment did not reach statistical significance for the primary or secondary outcome measures over the 28-day study period. However, the nasal mRNA from the group receiving 0.1 mg of digitoxin daily had a distinct distribution of global gene expression levels as compared with either the 0.05-mg dose or placebo treatment. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment, and mucus hypersecretion. CONCLUSIONS: At a dose of 0.1 mg daily for 28 days, digitoxin was safe for adults with CF lung disease, but it did not achieve a significant decrease in sputum inflammatory markers. Clinical trial registered with www.clinicaltrials.gov (NCT00782288).

Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases.

Acting on a broad spectrum of extracellular, intracellular, and membrane-associated substrates, the matrix metalloproteinases (MMPs) are critical to the biological processes of organisms; when aberrantly expressed, many pathological conditions may be born or exacerbated. The prospect of MMP inhibition for therapeutic benefit in cancer, cardiovascular disease, and stroke is reviewed here. MMP inhibitor (MMPI) development constitutes an important branch of research in both academic and industrial settings and advances our knowledge on the structure-function relationship of MMPs. Targeting MMPs in disease treatment is complicated by the fact that MMPs are indispensable for normal development and physiology and by their multi-functionality, possible functional redundancy or contradiction, and context-dependent expression and activity. This complexity was revealed by previous efforts to inhibit MMP activity in the treatment of cancer patients that yielded unsatisfactory results. This review focuses on MMPI development since the late 90s, in terms of natural products and their derivatives, and synthetic compounds of low molecular mass incorporating specific zinc-binding groups (ZBGs). A few polyphenols and flavonoids that exhibit MMPI activities may have chemopreventive and neuro- and cardiovascular-protective effects. A new generation of potent and selective MMPIs with novel ZBGs and inhibition mechanisms have been designed, synthesized, and tested. Although only one collagenase inhibitor (Periostat, doxycycline hyclate) has been approved by the Food and Drug Administration as a drug for the treatment of periodontal disease, new hope is emerging in the form of natural and synthetic MMPIs for the prevention and treatment of stroke, cardiovascular disease, cancer, and other medical conditions.

Direct interactions between ENaC gamma subunit and ClCN2 in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a lethal disease caused by mutations in the chloride channel CFTR gene. The disease is characterized by decreased chloride secretion and unregulated sodium absorption through the epithelial sodium channel (ENaC) in the airway epithelium and other affected organs. We hypothesize that a non-CFTR alternative chloride channel ClCN2 can be activated to negatively regulate ENaC in CF epithelial cell cultures. We identified a novel interaction between ClCN2 and the ENaCγ subunit in CF airway epithelial cells and show that the upregulation of ClCN2 leads to decreased expression of ENaCγ via a K63 ubiquitination mechanism. These regulatory effects of ClCN2 on ENaCγ appear to be dependent on the CBS-1 domain located within the c-terminus of ClCN2, which is necessary for the targeting of ClCN2 to the apical surface. In sum, these results suggest the ability of ClCN2 to negatively regulate sodium absorption through ENaC, supporting its role as a therapeutic target for the treatment of CF.

Regulation of nasal airway homeostasis and inflammation in mice by SHP-1 and Th2/Th1 signaling pathways.

Allergic rhinitis is a chronic inflammatory disease orchestrated by Th2 lymphocytes. Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 is known to be a negative regulator in the IL-4α/STAT-6 signaling pathway of the lung. However, the role of SHP-1 enzyme and its functional relationship with Th2 and Th1 cytokines are not known in the nasal airway. In this study, we aimed to study the nasal inflammation as a result of SHP-1 deficiency in viable motheaten (mev) mice and to investigate the molecular mechanisms involved. Cytology, histology, and expression of cytokines and chemokines were analyzed to define the nature of the nasal inflammation. Targeted gene depletion of Th1 (IFN-γ) and Th2 (IL-4 and IL-13) cytokines was used to identify the critical pathways involved. Matrix metalloproteinases (MMPs) were studied to demonstrate the clearance mechanism of recruited inflammatory cells into the nasal airway. We showed here that mev mice had a spontaneous allergic rhinitis-like inflammation with eosinophilia, mucus metaplasia, up-regulation of Th2 cytokines (IL-4 and IL-13), chemokines (eotaxin), and MMPs. All of these inflammatory mediators were clearly counter-regulated by Th2 and Th1 cytokines. Deletion of IFN-γ gene induced a strong Th2-skewed inflammation with transepithelial migration of the inflammatory cells. These findings suggest that SHP-1 enzyme and Th2/Th1 paradigm may play a critical role in the maintenance of nasal immune homeostasis and in the regulation of allergic rhinitis.

Interference with ubiquitination in CFTR modifies stability of core glycosylated and cell surface pools.

It is recognized that both wild-type and mutant CFTR proteins undergo ubiquitination at multiple lysines in the proteins and in one or more subcellular locations. We hypothesized that ubiquitin is added to specific sites in wild-type CFTR to stabilize it and at other sites to signal for proteolysis. Mass spectrometric analysis of wild-type CFTR identified ubiquitinated lysines 68, 710, 716, 1041, and 1080. We demonstrate that the ubiquitinated K710, K716, and K1041 residues stabilize wild-type CFTR, protecting it from proteolysis. The polyubiquitin linkage is predominantly K63. N-tail mutants, K14R and K68R, lead to increased mature band CCFTR, which can be augmented by proteasomal (but not lysosomal) inhibition, allowing trafficking to the surface. The amount of CFTR in the K1041R mutant was drastically reduced and consisted of bands A/B, suggesting that the site in transmembrane 10 (TM10) was critical to further processing beyond the proteasome. The K1218R mutant increases total and cell surface CFTR, which is further accumulated by proteasomal and lysosomal inhibition. Thus, ubiquitination at residue 1218 may destabilize wild-type CFTR in both the endoplasmic reticulum (ER) and recycling pools. Small molecules targeting the region of residue 1218 to block ubiquitination or to preserving structure at residues 710 to 716 might be protein sparing for some forms of cystic fibrosis.

Extracellular matrix protein lumican regulates inflammation in a mouse model of colitis.

BACKGROUND: Abnormal innate immune response contributes to inflammatory bowel disease (IBD) and experimental mouse colitis. Colitis studies have focused primarily on key regulators of innate immunity, like pathogen recognition receptors and cytoplasmic mediators. Extracellular matrix (ECM) proteins are emerging as modulators of inflammatory responses by virtue of their interactions with pathogen-associated molecular patterns (PAMPs), cytokines, growth factors, receptors, and ECM fragments that mimic pathogens or cytokines. The ECM proteins have not been investigated in IBD at great depth from this standpoint. We have shown previously that the ECM protein lumican modulates host sensing of bacterial lipopolysaccharides (LPS) by Toll-like receptor (TLR) 4, and neutrophil chemotaxis via integrins. METHODS: Here we investigated the role of lumican in the development of colitis mediated by intrarectal administration of the hapten 2-4-5, trinitrobenzene sulfonic acid (TNBS) in Lum(+/+) and Lum(-/-) mice. RESULTS: The TNBS treated Lum(+/+) mouse colons showed marked increases in CXCL1, tumor necrosis factor alpha (TNF-α), and neutrophil infiltration, whereas these responses were significantly dampened in the Lum(-/-) mice. The nuclear factor kappa B (NF-κB) transcription factor, known to regulate inflammatory genes, showed a robust increase after TNBS treatment in Lum(+/+) but not in Lum(-/-) colons. Also, nuclear translocation of NF-κB was delayed in LPS stimulated Lum(-/-) primary peritoneal macrophages. CONCLUSIONS: The Lum(-/-) mice have low innate immune and inflammatory responses, but more severe body weight loss and tissue damage, a phenomenon seen in the innate immune impaired Tlr4(-/-) and MyD88(-/-) mice. Therefore, lumican promotes intestinal homeostasis by aiding innate immune and inflammatory responses that are beneficial in the early stages of colitis.

Subnormal cytokine profile in the tear fluid of keratoconus patients.

Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1ß, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-γ, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-α were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-α, IFN-γ, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-α and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-α, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.

Protein Signatures in Human MDA-MB-231 Breast Cancer Cells Indicating a More Invasive Phenotype Following Knockdown of Human Endometase/Matrilysin-2 by siRNA.

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a putative biomarker for carcinomas of breast, prostate, and other cancers of epithelial origin. MMP-26 expression was silenced using small interfering RNA (siRNA) in the human breast cancer cell line MDA-MB-231. Immunological and proteomics approaches, including two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry, were employed to identify differential protein expression in MMP-26 knockdown cells. A comparison of the protein expression profiles of control and MMP-26 knockdown cells revealed nine differentially regulated proteins. Five of the proteins (heat shock protein 90, glucose-regulated protein 78 (GRP78), annexin V, tropomyosin, and peroxiredoxin II) were up-regulated, while alpha-tubulin, cystatin SA-III, breast cancer metastasis suppressor 1 (BRMS1) and beta-actin were down-regulated. This decrease of BRMS1 expression is concomitant with an increase of invasion through matrix-coated membranes. These results suggest an important role for MMP-26 in the regulation of proteins involved in invasive and metastatic breast cancers.

A novel antimicrobial peptidoglycan recognition protein in the cornea.

PURPOSE: In an earlier gene expression study, the authors identified a novel antimicrobial gene, Peptidoglycan recognition protein 1 (Pglyrp1), in the mouse cornea. Here the expression of the Pglyrp1 transcript and the encoded protein, PGLYRP1, in the cornea was investigated. The role of PGLYRP1 in the cornea was further investigated using wild-type and Pglyrp1-deficient mice. This is the first report of this antimicrobial protein in the cornea. METHODS: PGLYRP1 was detected in the cornea and was further localized to the epithelium by immunohistology, confocal microscopy, immunoblotting, and real-time PCR. The role of PGLYRP1 in the cornea was investigated by comparing the response of wild-type and Pglyrp1(-/-) mice to corneal epithelial wounds and Pseudomonas aeruginosa-mediated corneal infections. The antibacterial effects of corneal PGLYRP1 were assayed by measuring bacterial growth in vitro, in the presence of wild-type corneal epithelial extracts, before and after antibody-mediated blocking of PGLYRP1. RESULTS: PGLYRP1 is expressed at high levels in the mouse corneal epithelium. PGLYRP1 was localized to the mouse corneal epithelium and the human corneal epithelium. The Pglyrp1(-/-) mouse shows delayed healing and poor clearing of bacterial keratitis; in vitro its epithelial protein extract shows reduced bacteriostatic activity compared with wild-type mice. CONCLUSIONS: PGLYRP1 is a novel antimicrobial protein of the corneal epithelium and protects the ocular surface from bacterial infections.

Extracellular matrix lumican deposited on the surface of neutrophils promotes migration by binding to beta2 integrin.

During inflammation, circulating polymorphonuclear neutrophils (PMNs) receive signals to cross the endothelial barrier and migrate through the extracellular matrix (ECM) to reach the injured site. Migration requires complex and poorly understood interactions of chemokines, chemokine receptors, ECM molecules, integrins, and other receptors. Here we show that the ECM protein lumican regulates PMN migration through interactions with specific integrin receptors. Lumican-deficient (Lum(-/-)) mice manifest connective tissue defects, impaired innate immune response, and poor wound healing with reduced PMN infiltration. Lum(-/-) PMNs exhibit poor chemotactic migration that is restored with exogenous recombinant lumican and inhibited by anti-lumican antibody, confirming a role for lumican in PMN migration. Treatment of PMNs with antibodies that block beta(2), beta(1), and alpha(M) integrin subunits inhibits lumican-mediated migration. Furthermore, immunohistochemical and biochemical approaches indicate binding of lumican to beta(2), alpha(M), and alpha(L) integrin subunits. Thus, lumican may regulate PMN migration mediated by MAC-1 (alpha(M)/beta(2)) and LFA-1 (alpha(L)/beta(2)), the two major PMN surface integrins. We detected lumican on the surface of peritoneal PMNs and not bone marrow or peripheral blood PMNs. This suggests that PMNs must acquire lumican during or after crossing the endothelial barrier as they exit circulation. We also found that peritoneal PMNs do not express lumican, whereas endothelial cells do. Taken together these observations suggest a novel endothelial lumican-mediated paracrine regulation of neutrophils early on in their migration path.

Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor.

The identification of novel biomarkers for early prostate cancer diagnosis is highly important because early detection and treatment are critical for the medical management of patients. Disruption in the continuity of both the basal cell layer and basement membrane is essential for the progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to invasive adenocarcinoma in human prostate. The molecules involved in the conversion to an invasive phenotype are the subject of intense scrutiny. We have previously reported that matrix metalloproteinase-26 (MMP-26) promotes the invasion of human prostate cancer cells via the cleavage of basement membrane proteins and by activating the zymogen form of MMP-9. Furthermore, we have found that tissue inhibitor of metalloproteinases-4 (TIMP-4) is the most potent endogenous inhibitor of MMP-26. Here we demonstrate higher (p<0.0001) MMP-26 and TIMP-4 expression in HGPIN and cancer, compared to non-neoplastic acini. Their expression levels are highest in HGPIN, but decline in invasive cancer (p<0.001 for each) in the same tissues. Immunohistochemical staining of serial prostate cancer tissue sections suggests colocalization of MMP-26 and TIMP-4. The present study indicates that MMP-26 and TIMP-4 may play an integral role during the conversion of HGPIN to invasive cancer and may also serve as markers for early prostate cancer diagnosis.

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling.

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.