Meprin Metalloproteases Research Articles

Let It Flow: Morpholino Knockdown in Zebrafish Embryos Reveals a Pro-Angiogenic Effect of the Metalloprotease Meprin α2

BackgroundMeprin metalloproteases are thought to be involved in basic physiological functions such as cell proliferation and tissue differentiation. However, the specific functions of these enzymes are still ambiguous, although a variety of growth factors and structural proteins have been identified as meprin substrates. The discovery of meprins α1, α2 and β in teleost fish provided the basis for uncovering their physiological functions by gene silencing in vivo.Methodology/Principal FindingsA Morpholino knockdown in zebrafish embryos targeting meprin α1 and β mRNA caused defects in general tissue differentiation. But meprin α2 morphants were affected more specifically and showed severe failures in the formation of the vascular system provoking the hypothesis of a pro-angiogenic effect. The blood circulation was largely diminished resulting in erythrocyte accumulation. These phenotypes mimic a previously described VEGF-A morphant, revealing a possible role of meprin α in VEGF-A activation. Indeed, human recombinant meprin α processed the vascular endothelial growth factor-A (VEGF-A) specifically, revealing the same cleavage products detectable for VEGF from zebrafish whole lysate.Conclusions/SignificanceOur results demonstrate that meprin metalloproteases are important for cell differentiation and proliferation already during embryogenesis, predominantly by the activation of growth factors. Thus, we conclude that meprins play a significant role in VEGF-A processing, subsequently regulating angiogenesis. Therefore, meprin α might be a new therapeutic target in cardiovascular diseases or in tumor growth inhibition.

Meprin metalloproteases are involved in monocyte trans-extracellular matrix migration and meprin deficiency aggravates LPS-induced septic shock (134.56)

Abstract Meprins are metalloproteases first discovered in kidney and intestinal epithelial cells, and have been implicated in autoimmune diseases. We have shown that homeostasis of monocytes is compromised in meprin alpha/beta null (alpha-/-/beta-/-) mice. However, the mechanisms responsible for the involvement of meprins in the immune and hematological system remain uncharacterized. We report here that human monocytes expressed meprins, and the expression of meprins was down-regulated in mature macrophages and dendritic cells but not their immature counterparts. We further show that bone marrow monocytes and peritoneal macrophages from meprin-deficient mice had decreased efficiency in migration through extracellular matrix (ECM) compared to those from the wild-type mice in Boyden assays without marked defects in cell mobility in the absence of ECM. In a sepsis shock model, meprin-deficient mice exhibited more severe hypothermia than the wild-type mice in response to peritoneally injected lipopolysaccharide (LPS), and the prevalence of blood monocytes in the merpin-deficient mice were only 25% of that in the wild-type mice, with compensatory increase of lymphocytes. While meprin-deficient mice in the steady state had moderately lower blood levels of IL-12, MCP-1 and IL-6 than the wild-type mice, LPS-challenged meprin-deficient mice exhibited a drastic elevation of the above inflammatory cytokines in comparison to the wild-type mice, with comparable blood concentrations of IFN-gamma and IL-10 between the two mice. These results indicate that meprin metalloproteases are required for efficient trans-ECM migration of monocytes, and meprin deficiency is associated with defects in monocyte functions which have pathological consequences.

Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge

Meprin metalloproteases, composed of alpha and/or beta subunits, consist of membrane-bound and secreted forms that are abundantly expressed in proximal tubules of the kidney as well as secreted into the urinary tract. Previous studies indicated that meprin metalloproteases play a role in pathological conditions such as ischemic acute renal failure and urinary tract infection. The aim of this work was to examine the role of meprins in endotoxemic acute renal failure using meprin alpha knockout (alphaKO), meprin beta knockout (betaKO), and wild-type (WT) mice. Differences among the responses of the genotypes were observed as early as 1 h after challenge with 2.5 mg/kg ip Escherichia coli LPS, establishing roles for meprins in the endotoxemic response. Meprin alphaKO mice displayed lower blood urea nitrogen levels and decreased nitric oxide levels, indicative of a decreased systemic response to LPS compared with WT and meprin betaKO mice. Serum cytokine profiles showed lower levels of IL-1beta and TNF-alpha in the meprin alphaKO mice within 3 h after LPS challenge and confirmed a role for meprins in the early phases of the host response. Meprin alphaKO mice were also hyporesponsive to LPS administered to the bladder, exhibiting significantly less bladder edema, leukocyte infiltration, and bladder permeability than WT mice. These data indicate that meprin A contributes to the renal and urogenital pathogenesis of endotoxicity.

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Meprin metalloproteases play a role in host response to urinary tract infection

Meprin metalloproteases play a role in host response to urinary tract infection

Two α subunits and one β subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

Meprin metalloproteases confer resistance to urinary tract infections

Meprins are membrane-bound and secreted zinc metalloproteases that are expressed by kidney proximal tubules, intestinal epithelial cells, and leukocytes. They are found in the urinary tract, the intestinal lumen, and at sites of inflammation. The secreted form of meprin, composed of alpha subunits, is a large multimer capable of degrading bacterial proteins and shedding proteins from the cell surface. It was hypothesized that high levels of urinary meprin confer resistance to urinary tract infections (UTIs). Urine samples from healthy and UTI infected women were collected for analysis. Quantitative Western blotting showed that 89% of healthy women had low or undetectable levels of urinary meprin. In contrast, the majority (86%) of women suffering from an acute UTI had high levels of meprin protein in the urine. To examine susceptibility of different meprin genotypes to UTI, both wild-type and meprin alpha knockout mice were infected with Escherichia coli (E. coli) via transurethral catheterization. Bacterial counts in bladder show that meprin alpha knockout mice have 50 times greater number of E. coli versus wild-type. In addition, kidney bacterial counts in meprin alpha knockout mice were 13 fold greater in comparison to wild-type. These data indicate that meprin expression confers resistance to infections of the urinary tract. (NIH Grant DK 19691)

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity

The protease domains of the evolutionarily related alpha and beta subunits of meprin metalloproteases are approximately 55% identical at the amino acid level; however, their substrate and peptide bond specificities differ markedly. The meprin beta subunit favors acidic residues proximal to the scissile bond, while the alpha subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin beta, while it is not hydrolyzed by meprin alpha. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin alpha protease to cleave gastrin. The meprin alphaY199K mutant was most effective; the corresponding mutation of meprin betaK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin alphaTyr-199/betaLys-185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases.