Extended Data Fig. 6: TRPM7-mediated Ca2 + /Zn2+ signaling regulates Mmp2 transcription.
From: The TRPM7 chanzyme in smooth muscle cells drives abdominal aortic aneurysm in mice
(a) Pharmacology of TRPM7 chanzyme. (b) Gelatin zymography of WT cells pretreated for 1-hour with NS8593 (10 μM), BAPTA-AM (10 μM) or TPEN (10 μM), followed by Ang II stimulation (1 μM, 24 h). n = 3. Scale bar=200μm. (c) MMP14 transcripts in WT cells treated as in Fig. 4d. n = 5-6. (d) MMP14 transcripts in WT cells treated as in Fig. 4g. n = 4-6. (e) MMP2 mRNA in WT cells adenovirally transduced as indicated. Unpaired 2-tailed Student’s t test was performed (n = 9). (f) MMP2 mRNA in WT cells pretreated with PF431396 (10 μM), followed by naltriben (20 μM, 24 h) or Ang II stimulation. n = 6. (g) MMP14 mRNA in WT cells treated as in Fig. 4j. n = 6. (h) Adenoviral transduction of WT cells with WT or CA mutant of CnA. n = 6. (i) Nfatc2 (NFAT1), Nfatc1 (NFAT2), Nfatc4 (NFAT3), Nfatc3 (NFAT4) and Nfat5 (NFAT5) mRNA in aortic media from Trpm7sm-WT mice. n = 3. (j-k) MMP2 mRNA in WT cells. Adenovirus carrying target gene was transduced (MOI: 50) for 48-hours to measure MMP2 mRNA. n = 4-6. (l) Gelatin zymography of WT cells pretreated with NS8593 (10 μM), CsA (1 μM), CREB inhibitor 666-15 (1 μM) or MTF1 inhibitor APTO-253 (0.5 μM), followed by Ang II treatment. n = 3. Scale bar=200μm. (m) The schematic diagram depicting the predicted Mmp2 promoter and the truncated mutants. The 2010 base pairs upstream of Mmp2 start codon were predicted as the promoter. CREB or MTF1 binding sites were predicted using JASPAR (https://jaspar.genereg.net/) & PROMO(https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) tools. 9 CREB binding sites (the consensus binding sequence is TGACGT(CA)) were retrieved, including #1 (-1905 ~ -1898), #2 (-1795 ~ -1788), #3 (-1471 ~ -1464), #4 (-1351 ~ -1344), #5 (-959 ~ -952), #6 (-635 ~ -628), #7 (-351 ~ -345), #8 (-280 ~ -274), #9 (-37 ~ -30 or -28 ~ -21). No predicted MTF1 binding sites were retrieved. 3 truncated Mmp2 promoter mutants were generated: Δ523bp (-2010 ~ -1488) in mutant-1 to remove #1, 2 binding sites, Δ1025bp (-2010 ~ -986) in mutant-2 to remove #1, 2, 3, 4, or Δ1654bp (-2010 ~ -357) in mutant-3 to remove #1, 2, 3, 4, 5, 6 binding sites. The differences were determined by two-way ANOVA (Figs. 6c,d) or one-way ANOVA (Figs. 6f–h, j), followed by Tukey’s post-hoc test.
