MMPs Regulate both Development and Immunity in the Tribolium Model Insect Eileen Knorr, Henrike Schmidtberg, Andreas Vilcinskas, Boran Altincicek* Interdisciplinary Research Center, Institute of Phytopathology and Applied Zoology, Justus-Liebig-University of Giessen, Giessen, Germany Abstract Background: Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. Methodology/Principal Findings: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19. Conclusions/Significance: This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs. Citation: Knorr E, Schmidtberg H, Vilcinskas A, Altincicek B (2009) MMPs Regulate both Development and Immunity in the Tribolium Model Insect. PLoS ONE 4(3): e4751. doi:10.1371/journal.pone.0004751 Editor: Eleftherios Mylonakis, Massachusetts General Hospital, United States of America Received November 28, 2008; Accepted February 5, 2009; Published March 9, 2009 Copyright: 2009 Knorr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This project was supported by a grant (AL 902/2-1) to B. A. from the Deutsche Forschungsgemeinschaft (Bonn, Germany). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Boran.Altincicek@agrar.uni-giessen.de Introduction Matrix metalloproteinases (MMPs) are evolutionarily conserved but differ in gene family member number in animal species ranging from Cnidaria to Vertebrata. Humans have, for example, 22 different homologs [1] whereas the fruit fly Drosophila melanogaster has only two homologs [2]. Each of the vertebrate MMPs has distinct but overlapping functions and substrate specificities, and together they can cleave numerous extracellular substrates, including virtually all extracellular matrix proteins [3]. They are involved in mammalian physiological processes such as development, wound healing, and inflammation as well as in pathological states including cancers, rheumatism and osteoar- thritis [1–6]. Confirming their importance MMPs are tightly regulated at transcriptional and posttranslational levels. Proteol- ysis, allosteric interactions, and oxidative modification have been demonstrated to control MMP activities in the microenvironment of cells and tissues [7–8]. Moreover, specific physiological MMP- inhibitors have been identified including tissue inhibitor of metalloproteinases (TIMP) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK). TIMPs have conserved cysteine residues forming disulfide bridges [9–11] and besides inhibiting MMPs, mammalian TIMPs have also been implicated in direct regulation of cell growth and apoptosis by interacting with the cell surface receptors integrin a3b1 and CD63 [11]. RECK encodes a glycosylphosphatidylinositol (GPI)-anchored glycoprotein contain- ing multiple serine protease inhibitor-like motifs that negatively regulates MMPs in vivo [12,13]. As their name implies, MMPs were originally described as proteinases capable of degrading extracellular matrix proteins such as collagens, elastin, proteoglycans, and laminins. However, recent studies have further revealed that MMPs exhibit pleiotrophic roles by both activating or inactivating other proteinases, proteinase inhibitors, clotting factors, chemotactic molecules, latent growth factors, growth factor–binding proteins, antimicrobial peptides, and cell-surface receptors [2,7,8]. They are multifunctional effectors with dual promoting and inhibiting activities on numerous physiological processes such as tissue growth and PLoS ONE | www.plosone.org 1 March 2009 | Volume 4 | Issue 3 | e4751