An old dog's new tricks: iMMP‐3 Processes Nuclear Matrix and Converts Heterochromatin Proteins Leading to Transcriptional Promotion of HSP genes

Matrix Metalloproteinases (MMP) proteolytically process extracellular proteins; however, we have characterized a key role for intracellular MMP (iMMP) in gene transcription. We examined the hypothesis that nuclear architecture in cells regulates gene expression and may be modulated by iMMP‐3. Lamin is nuclear matrix lining inside of the nuclear membrane, and determining the nuclear shape and mechanical stability, chromatin organization, and transcription while heterochromatin protein 1 (HP1) anchors genomic DNA to lamin, and keeps the repressed status of transcription. We clarified that iMMP‐3 promotes cleavage of lamin A and conversion of HP1 leading to transcription of heat shock protein gene HSP70B’ and major alterations across the transcriptome . In addition, active iMMP‐3 promoted loss of nuclear size and formation of CLAGS (cleaved lamin aggregation speckles). Such processing of the nuclear matrix, chromatin conversion, and HSP gene expression promoted by iMMP‐3 may be influential in tumorigenesis, inflammatory diseases, and aging. Support or Funding Information This work was supported by a Joint Center for Radiation Therapy (JCRT) grant at Harvard Medical School 2015 (to T.E.), by Takeda Science Foundation Incentive Grant for Medical Research (to T.E.), by Ministry of Education, Culture, Sports, Science and Technology (MEXT)‐Japan Health and Labor Sciences Research Grants for Research on Publicly Essential Drugs and Medical Devices 22‐008 (to T.E.), by Japan Society for Promotion of Science (JSPS) Kakenhi Grant‐in‐Aid for Young Scientists B 20791378 (to T.E.), by National Institute of Health (NIH) research grants RO‐1CA047407, R01CA119045 and RO‐1CA094397 (to S.K.C.). 4 Proteolytic processing of Lamin A by iMMP‐3(A) Schemes of plasmid constructs expressing active iMMP3 (pACT.iMMP3) and catalytically dead iMMP3 (pCATdead‐H218/228R). (B) An experimental model of lamin A processing by active iMMP3. Lamin A is composed of two Globular domains (Glob.) that are connected by a rod domain (top). An anti‐Lamin A/C antibody #2023 can detect native and cleaved lamin A/C. The PEX domain of iMMP‐3 can capture lamin A/C, but the PrePro domain inhibit the own protease activity (middle). Active iMMP‐3 or the CAT domain can cleave lamin A (bottom). The cleaved lamin can be recognized by the antibody #2023 in Western blotting, and by the antibody #PA5‐17838 in immunofluorescences. The cleaved forms of lamin A/C may aggregate and show speckles. (C) Western blot analysis of cleaved and native lamin A/C. pACT.iMMP3 and pCAT were transfected into HeLa cells. Asterisks indicate native lamin A/C. An arrowhead indicates cleaved lamin. Active iMMP3 (ACT) and the catalytic domain (CAT) were indicated with an arrowhead and an arrow, respectively. The active iMMP3 was partially fragmented to CAT (an arrow). (D) Fluorescent microscopy analyses of MMP3‐gfp and endogenous cleaved lamin. MMP3‐gfp was expressed with the signal peptide in COS7 cells. NF45/ILF2 is a co‐factor of iMMP3 and marking nucleus. The antibody #PA5‐17838 was used. Cell#1. arrows indicate colocalization of cleaved lamin and MMP3‐gfp. Cell#2, cleaved lamin and MMP3‐gfp were localized to the intra‐nuclear periphery as well as cytoplasm. The boxed parts in the lower magnification images shown on the bottom were enlarged and shown as cell#2. (E) Fluorescent microscopy analyses of iMMP‐3 mutants and gfp‐Lamin A. iMMP3/PrePro. ACT, CAT, and CAT‐dead were co‐overexpressed with gfp‐Lamin A and RBBP4‐ha3 in HeLa cells. RBBP4 is a co‐factor of iMMP3 as well as a marker of the nucleus. Top raw, an iMMP3/PrePro positive cell. Red arrowheads indicate wavy nuclear lamina colocalized by iMMP‐3 and RBBP4 Second row, an ACT.iMMP3 positive cell. Arrows indicate co‐localization of the ACT and gfp‐lamin A. Third raw, a CAT domain positive cell. Arrows indicate speckles of gfp‐Lamin A and CAT that are physically dose to each other. Bottom row, a CAT‐dead positive cell. Arrowheads indicate gfp‐Lamin A and CAT‐dead iMMP3 co‐localization along the nuclear membrane. (F) Percentage of cells with lamin A speckles. n= 7 to 10. (G) Altered area of nuclei. n= 7 to 10.7 The iMMP model of chromatin transcription and lamin cleavageHeteroChromatin protein α (HP1α/CBX5) associates with repressed chromatin, repressive complex/factors (R), and nuclear matrix lamin (purple). iMMP3 (red) association with HP1α releases these repressive factors and RNA polymerase II (RNAP2/Polll) leading to promotion of HSP gene transcription. Activated iMMP3 can cleave lamin A leading to formation of CLAGS (cleaved lamin aggregated speckles). Chromatin decondensation (decond.) promoted by these roles for iMMP3 improve DNA accessibility of DNA binding factors (DBFs) such as HSF1.