Metalloproteinases in development and disease

University dissertation from Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Abstract: Metalloproteinases are Zn-dependent proteases composed of five superfamilies. One important subfamily is the matrix metalloproteinases (MMPs) that process various components of the extracellular matrix (ECM) and non-ECM proteins. They have been implicated in various physiological and pathologic conditions including cellular differentiation, tissue remodeling, wound healing, angiogenesis, and tumor invasion. To date, 24 different vertebrate MMPs have been identified, of which six are membrane bound. Membrane-type 1 matrix metalloproteinase (MT1-MMP), encoded by MMP14, was firstly identified in invasive tumor cells to serve as the receptor and activator of proMMP2. To investigate the biological functions of MT1-MMP, we generated Mmp14 knock-out mice by conventional gene targeting. Homozygous mutant mice had severe defects in skeletal develop-ment, lung and salivary gland branching morphogenesis, tooth eruption, and angiogenesis. The craniofacial, axial, and appendicular skeletons were severely affected. The null mice were growth retarded and died prematurely by 3-4 weeks of age. The hypertrophic zone in the growth plates was enlarged and the formation of secondary ossification centers was delayed. At postnatal day 18–20, the first molars of the Mmp14 null mice had apparently normal tooth crowns with normal dentin and enamel; however, the roots were truncated and the teeth had not yet erupted. Mmp14 null mice showed abnormal morphology and a reduction in epithelial branching of embryonic SMGs in vitro. Abnormal lung morphology in Mmp14 -/- mice arose after E16.5. There was defective septation and over-inflation of immature alveolar sacs in postnatal Mmp14 null mice. Mmp14 null mice show impaired angiogenesis stimulated by FGF-2 but not by VEGF, suggesting that the defective angiogenesis is not restricted to cartilage alone and the defective angiogenesis in Mmp14 knockout mice might be FGF signaling related. In null mice, activation of proMMP2 was impaired, suggesting that MT1-MMP is essential for its activation in vivo but activating proMMP2 is not the major biological function for MT1-MMP as Mmp2 null mice developed normally. ZMPSTE24 is a metalloproteinase on the endoplasmic reticulum and nuclear envelop with a HEXXH consensus sequence. It was predicted to participate in the proteolytic cleavage of farnesylated proteins with CAAX at C-terminus. Disruption of Zmpste24 in mice caused postnatal growth retardation and premature death in mice. Homozygous mutant mice displayed several abnormalities, including lipodystrophy, muscular dystrophy, alopecia, and osteoporosis. These alterations were similar to those observed in mice deficient for A-type lamin, a major component of the nuclear lamina, and recapitulated many of the phenotypes observed in patients suffering from congenital laminopathies and premature aging. In agreement with this finding, Zmpste24 null mice were defective in the proteolytic processing of prelamin A. The deficiency in prelamin A maturation led to abnormal nuclear envelope architecture, increased genomic instability and accelerated cellular senescence that probably underlies many of the phenotypes observed in both progerial patients and other laminopathies. It may affect the aging process by affecting gene expression and DNA-damage checkpoint response. These results indicated that proper processing of nuclear lamin A is critical for the integrity of nuclear envelope and genomic stability. Loss of the functional lamin A may lead increased DNA damage, compromised the DNA-repair, thus affect genomic maintenance. Furthermore, the identification of prelamin A as a specific substrate for ZMPSTE24 demonstrates the usefulness of genetic approaches for identifying the in vivo substrates of proteolytic enzymes.

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