Role of Coronin 1B in PDGF-Induced Migration of Vascular Smooth Muscle Cells

The type I subclass of Coronins, a family of actin binding proteins, regulates various actin dependent cellular processes including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined

Angiotensin II Requires Zinc and Downregulation of the Zinc Transporters ZnT3 and ZnT10 to Induce Senescence of Vascular Smooth Muscle Cells

Senescence, a hallmark of mammalian aging, is associated with the onset and progression of cardiovascular disease. Angiotensin II (Ang II) signaling and zinc homeostasis dysfunction are increased with age and are linked to cardiovascular disease, but the relationship among these processes has not been investigated. We used a model of cellular senescence induced by Ang II in vascular smooth muscle cells (VSMCs) to explore the role of zinc in vascular dysfunction. We found that Ang II-induced senescence is a zinc-dependent pathway mediated by the downregulation of the zinc transporters ZnT3 and ZnT10, which work to reduce cytosolic zinc. Zinc mimics Ang II by increasing reactive oxygen species (ROS), activating NADPH oxidase activity and Akt, and by downregulating ZnT3 and ZnT10 and inducing senescence. Zinc increases Ang II-induced senescence, while the zinc chelator TPEN, as well as overexpression of ZnT3 or ZnT10, decreases ROS and prevents senescence. Using HEK293 cells, we found that ZnT10 localizes in recycling endosomes and transports zinc into vesicles to prevent zinc toxicity. Zinc and ZnT3/ZnT10 downregulation induces senescence by decreasing the expression of catalase. Consistently, ZnT3 and ZnT10 downregulation by siRNA increases ROS while downregulation of catalase by siRNA induces senescence. Zinc, siZnT3 and siZnT10 downregulate catalase by a post-transcriptional mechanism mediated by decreased phosphorylation of ERK1/2. These data demonstrate that zinc homeostasis dysfunction by decreased expression of ZnT3 or ZnT10 promotes senescence and that Ang II-induced senescence is a zinc and ROS-dependent process. Our studies suggest that zinc might also affect other ROS-dependent processes induced by Ang II, such as hypertrophy and migration of smooth muscle cells

Model representing Ang II and zinc-dependent senescence mechanisms.

<p>Similar to Ang II, zinc activates NADPH oxidase activity, increases ROS levels and Akt phosphorylation and downregulates ZnT3, ZnT10 and catalase. However, Ang II-induced downregulation of catalase is mediated by an Akt-dependent transcriptional mechanism, while zinc-induced downregulation of catalase is mediated by an ERK1/2-dependent post-transcriptional mechanism. Downregulation of catalase further increases ROS that mediates senescence. Perpendicular red arrows indicate increased or reduced expression.</p

ZnT10 localizes and transports zinc into early/recycling endosomes in HEK293 cells.

<p>HEK293 cells transfected with ZnT10-myc alone (A) or together with ZnT3-GFP (B) were fixed in 4% PFA and analyzed by immunofluorescence with monoclonal antibodies against Tfr-R, EEA1 and CD63 (A) and polyclonal antibodies against myc (A and B). Images were acquired with a confocal microscope using a 63×/oil objective. C) HEK293 cells transiently transfected with ZnT10-myc and ZnT3-GFP were cross-linked with 1 mM DSP, lysed and Triton X-100 soluble extracts immunoprecipitated with monoclonal antibodies against myc. ZnT3 and ZnT10 interaction (lanes 6 and 7) was determined by western blots using polyclonal antibodies against myc and GFP. 10 µg of Triton X-100 soluble extracts were used as inputs (lanes 8–10). D and E) Control and HEK293 stable cells lines expressing ZnT10-myc were incubated with and without zinc and stained with Zinpyr-1. Fluorescence intensity was determined by confocal microscopy and quantified using MetaMorph software. F) Cell viability was determined by trypan-blue exclusion in HEK293 control and ZnT10-myc expressing cells after incubations with 100 µM zinc for 24 hrs. Bar = 10 µm in A, B and D.</p

Ang II downregulates the zinc transporters ZnT3 and ZnT10 to induce senescence.

<p>A) VSMCs incubated with 100 nM Ang II for three days in the presence or absence of 100 nM TPEN were incubated with Zinpyr-1 and imaged by confocal microscopy. B) Total mRNA was used to determine ZnT1 to ZnT10 expression levels by RT-PCR in VSMCs and mouse aorta using GAPDH as control. All primers were designed to recognize mouse and rat ZnTs, except for ZnT1 and ZnT3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033211#pone.0033211.s007" target="_blank">Table S1</a>). C) mRNA prepared from VSMCs treated for one to three days with 100 nM Ang II was used to determine expression of the indicated ZnTs by RT-PCR. Cells treated with siRNA to downregulate ZnT3 and ZnT10 were imaged with Zinpyr-1 (D and E) or processed for SA-β-gal staining (F and G) or western blot to determine p21 and p53 expression (H). siZnT5-treated cells were analyzed for SA-β-gal staining (F and G). I and J) Cells were treated with Ang II, zinc or both for three days and samples analyzed by RT-PCR and quantified respect to GAPDH control. Bar = 10 µm in A and D and 200 µm in F. * and ** represent p<0.05 and p<0.01, respectively.</p

Zinc is required for Ang II-induced senescence by a ROS-dependent pathway.

<p>A) Cells incubated with and without Ang II in the presence or absence of 50 µM zinc or 100 nM TPEN for 30 min were incubated with H₂DCFDA to determine ROS levels. SA-β-gal activity was determined after three (B–D) or five (E and F) days by counting SA-β-gal positive cells (C and F) or by quantitative luminescence (D). E and F) Cells were incubated with zinc, zinc plus 1 mM NAC or 50 µM H₂O₂ and senescence determined by SA-β-gal staining. Bar = 200 µm.</p

Catalase is downregulated by zinc by an Akt-independent ERK1/2-dependent post-transcriptional pathway.

<p>VSMCs treated with and without 1 mM NAC, 1 µM of the Akt inhibitor V tricibirine (TCN) (A and C) or transfected with FoxO1 <i>wt</i> or FoxO1-CA (B and C) were incubated with or without 50 µM zinc for three days and protein expression determined by western blots with the indicated antibodies. C) The effect of zinc was quantified for each condition independently, considering basal levels of catalase with or without NAC, TCN or FoxO1 overexpression as 1. Cells incubated for three days with 50 µM zinc or 2 µM of the ERK1/2 inhibitor PD98059 (PD) were processed for western blot to determine catalase expression (D and E) or for SA-β-gal staining (F and G). Bar = 100 µm in F. VSMCs treated with siZnT3 and siZnT10 (H and I) or transfected with ZnT3-myc or ZnT10-myc plasmids (J and K) were lysed and protein levels of p-ERK1/2 (I and K) and catalase (K) quantified by western blots. ** and ns denote p<0.01 and non-statistic differences, respectively.</p

Downregulation of catalase by zinc, Ang II, siZnT3 and siZnT10 mediates senescence in VSMCs.

<p>A) Cells incubated with and without 100 nM Ang II, zinc (25 or 50 µM) or 100 nM TPEN for five days were lysed and catalase expression determined by western blots. B and C) VSMCs incubated with and without 50 µM zinc for one, two or three days were lysed and protein expression of the indicated antioxidant enzymes determined by western blots and quantified after 3 days (C). D and E) Catalase expression was determined by western blot after ZnT3 and ZnT10 knock down by siRNA. F) Cells treated with siRNA to downregulate ZnT3, ZnT10 or catalase (Cat) were incubated with H₂DCFDA to determine ROS levels. SA-β-gal positive cells were counted in siControl and siCatalase-treated cells (G) and in catalase-transfected cells (H) after five days treatment with and without Ang II or zinc 50 µM. ** and ns denote p<0.01 and non-statistic differences, respectively.</p