Paralogues of Mmp11 and Timp4 Interact during the Development of the Myotendinous Junction in the Zebrafish Embryo

Abstract

The extracellular matrix (ECM) of the myotendinous junction (MTJ) undergoes dramatic physical and biochemical remodeling during the first 48 h of development in zebrafish, transforming from a rectangular fibronectin-dominated somite boundary to a chevron-shaped laminin-dominated MTJ. Matrix metalloproteinase 11 (Mmp11, a.k.a. Stromelysin-3) is both necessary and sufficient for the removal of fibronectin at the MTJ, but whether this protease acts directly on fibronectin and how its activity is regulated remain unknown. Using immunofluorescence, we show that both paralogues of Mmp11 accumulate at the MTJ during this time period, but with Mmp11a present early and later replaced by Mmp11b. Moreover, Mmp11a also accumulates intracellularly, associated with the Z-discs of sarcomeres within skeletal muscle cells. Using the epitope-mediated MMP activation (EMMA) assay, we show that despite having a weaker paired basic amino acid motif in its propeptide than Mmp11b, Mmp11a is activated by furin, but may also be activated by other mechanisms intracellularly. One or both paralogues of tissue inhibitors of metalloproteinase-4 (Timp4) are also present at the MTJ throughout this process, and yeast two-hybrid assays reveal distinct and specific interactions between various domains of these proteins. We propose a model in which Mmp11a activity is modulated (but not inhibited) by Timp4 during early MTJ remodeling, followed by a phase in which Mmp11b activity is both inhibited and spatially constrained by Timp4 in order to maintain the structural integrity of the mature MTJ.

The Roles of Matrix Metalloproteinases and Their Inhibitors in Human Diseases

“…In these cases, only the C terminus is involved, leaving the N terminus free to associate with a second MMP molecule [ 10 , 83 ]. TIMPs are also involved in MMP activation and possible modulation through mediating interactions of active MMPs with specific substrates [ 84 ]. In vivo studies using MMP inhibitors and MMP knockout mice indicate that MMPs have essential roles in infection and host defense [ 85 ].…”

Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos

“…Mmp13a is the first protein identified in the upstream activation pathway of zHE1, but it is not yet clear if Mmp13a is activating zHE1 directly or if it is further upstream. Delving into the specific biochemistry of zHE1 activation will require an antibody against zHE1 or some other technique to measure metalloprotease activation such as the Epitope-Mediated MMP Activation assay [33,61]. Identification of Mmp13a as the putative activator of zHE1 has, in a sense, moved the goalposts, as now we must ask how Mmp13a is being activated and regulated in the hatching gland.…”

“…The matrix metalloproteinases (MMPs) are a highly conserved family of secreted, zinc-dependent endopeptidases that are best known for their ability to degrade and remodel the extracellular matrix (ECM) [28,29]. More recently, it has become clear that ECM remodeling is only one facet of a complex suite of the biological functions in which these proteases play a central role [30][31][32][33]. There are 25 genes encoding MMPs in the zebrafish genome [34].…”

“…This number may inflate the apparent degree of diversity of MMPs in fish, as many are duplicates that arose from the ancestral teleost genome duplication [35,36]. However, it is becoming clear that neo-or sub-functionalization has occurred as these paralogues diverged [33]. MMP1, -8 and -13 are the "collagenases" found in mammalian genomes, and all three of these MMPs can efficiently degrade fibrillar collagens [37].…”

Development and maintenance of tendons and ligaments

“…During MTJ development in vertebrates, a careful balance between ECM deposition and remodeling is maintained in response to mechanical signals. This balance is achieved through reciprocal interactions between structural proteins, such as laminin and collagens, and matrix metalloproteinases (Brown, 2000;Matchett et al, 2019;Zhang et al, 2014). As previously discussed, both molecular and mechanical factors originating from the muscle play a vital role in inducing tendon progenitors in the axial skeleton and promoting tendon cell maturation in the limb and craniofacial complex.…”