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Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

Bottom Line: Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects.We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

ABSTRACT
Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

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TRAF4–Hic-5 interactions affect endothelial cell migration. (A) HUVEC were transfected with siRNA against either TRAF4 or Hic-5, and migration across filters in response to a VEGF gradient was measured. Immunoblots show protein levels 48 h after transfection. Control siRNA was against luciferase. 100 μM MnTMPyP was present only during the 16-h migration period. Knockdown of Hic-5 or TRAF4 or antioxidant treatment decreased VEGF-induced migration of HUVEC. hpf, high-powered field. (B) Phoenix-293 cells were cotransfected with full-length TRAF4 and Flag-tagged Hic-5 truncations as indicated. Immunoblots of lysates before TRAF4 immunoprecipitation are shown as input. Cartoon below diagrams Hic-5 truncations showing NH2-terminal Flag tag, three LD motifs (shaded), and four COOH-terminal LIM domains (open). Only full-length Flag–Hic-5 containing LIM 4 coprecipitated with TRAF4 (first lane). (C) Phoenix-293 cells were cotransfected with full-length TRAF4 and full-length Flag–Hic-5 (FL), Flag–Hic-5(329–444) (LIM3,4), or Flag–Hic-5(388–444) (LIM4). Lysates before TRAF4 immunoprecipitation were immunoblotted for Hic-5 and reprobed for TRAF4 to show input. Top panel shows coprecipitation of full-length Flag–Hic-5 and Flag–Hic-5(329–444) but not Flag–Hic-5(388–444) with TRAF4. (D) Phoenix-293 cells were cotransfected with full-length Flag–Hic-5 and either Flag-TRAF4(1–260) (TRAF4-N) or Flag-TRAF4(261–470) (TRAF4-C). Lysate was immunoblotted with anti-Flag (bottom) to show Hic-5 and TRAF4 inputs simultaneously. After immunoprecipitation with anti–Hic-5, blots were probed with anti-Flag to demonstrate the recovery of Flag–TRAF4-C but not Flag–TRAF4-N. Ig heavy and light chains (HC and LC) are shown. (E) HUVEC were cotransfected with pEGFP and empty vector (pCIN), TRAF4-C, or Hic-5(LIM3,4). GFP-expressing cells migrating across 8-μm pore filters in response to a VEGF gradient is shown. Migration in response to VEGF was blocked by the coexpression of either TRAF4-C or Hic-5(LIM3,4). (A and E) *, P < 0.05 compared with no VEGF control; †, P < 0.05 compared with VEGF control. (F) Discrete DsRed-p47 dotlike structures characteristically appeared at the edges of lamellar protrusions (arrows). Bar, 20 μm. Cotransfection of HUVEC with TRAF4-C or knockdown of endogenous TRAF4 or Hic-5 decreased the number of cells (*, P < 0.05) with such DsRed-p47 structures. (G) HUVEC were cotransfected as in D and plated on fibronectin-coated etched coverslips. Phase-contrast and epifluorescent (green) images were obtained immediately after wounding and at 16 and 24 h after wounding. (H) Histogram represents mean speed of GFP-expressing cells migrating into the wound. Entry of GFP-expressing cells into the wound was decreased by the coexpression of TRAF4-C or Hic-5(LIM3,4). Non–GFP-expressing cells migrated into wounds at comparable speeds. Error bars represent SEM. *, P < 0.05 compared with vector alone.
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fig4: TRAF4–Hic-5 interactions affect endothelial cell migration. (A) HUVEC were transfected with siRNA against either TRAF4 or Hic-5, and migration across filters in response to a VEGF gradient was measured. Immunoblots show protein levels 48 h after transfection. Control siRNA was against luciferase. 100 μM MnTMPyP was present only during the 16-h migration period. Knockdown of Hic-5 or TRAF4 or antioxidant treatment decreased VEGF-induced migration of HUVEC. hpf, high-powered field. (B) Phoenix-293 cells were cotransfected with full-length TRAF4 and Flag-tagged Hic-5 truncations as indicated. Immunoblots of lysates before TRAF4 immunoprecipitation are shown as input. Cartoon below diagrams Hic-5 truncations showing NH2-terminal Flag tag, three LD motifs (shaded), and four COOH-terminal LIM domains (open). Only full-length Flag–Hic-5 containing LIM 4 coprecipitated with TRAF4 (first lane). (C) Phoenix-293 cells were cotransfected with full-length TRAF4 and full-length Flag–Hic-5 (FL), Flag–Hic-5(329–444) (LIM3,4), or Flag–Hic-5(388–444) (LIM4). Lysates before TRAF4 immunoprecipitation were immunoblotted for Hic-5 and reprobed for TRAF4 to show input. Top panel shows coprecipitation of full-length Flag–Hic-5 and Flag–Hic-5(329–444) but not Flag–Hic-5(388–444) with TRAF4. (D) Phoenix-293 cells were cotransfected with full-length Flag–Hic-5 and either Flag-TRAF4(1–260) (TRAF4-N) or Flag-TRAF4(261–470) (TRAF4-C). Lysate was immunoblotted with anti-Flag (bottom) to show Hic-5 and TRAF4 inputs simultaneously. After immunoprecipitation with anti–Hic-5, blots were probed with anti-Flag to demonstrate the recovery of Flag–TRAF4-C but not Flag–TRAF4-N. Ig heavy and light chains (HC and LC) are shown. (E) HUVEC were cotransfected with pEGFP and empty vector (pCIN), TRAF4-C, or Hic-5(LIM3,4). GFP-expressing cells migrating across 8-μm pore filters in response to a VEGF gradient is shown. Migration in response to VEGF was blocked by the coexpression of either TRAF4-C or Hic-5(LIM3,4). (A and E) *, P < 0.05 compared with no VEGF control; †, P < 0.05 compared with VEGF control. (F) Discrete DsRed-p47 dotlike structures characteristically appeared at the edges of lamellar protrusions (arrows). Bar, 20 μm. Cotransfection of HUVEC with TRAF4-C or knockdown of endogenous TRAF4 or Hic-5 decreased the number of cells (*, P < 0.05) with such DsRed-p47 structures. (G) HUVEC were cotransfected as in D and plated on fibronectin-coated etched coverslips. Phase-contrast and epifluorescent (green) images were obtained immediately after wounding and at 16 and 24 h after wounding. (H) Histogram represents mean speed of GFP-expressing cells migrating into the wound. Entry of GFP-expressing cells into the wound was decreased by the coexpression of TRAF4-C or Hic-5(LIM3,4). Non–GFP-expressing cells migrated into wounds at comparable speeds. Error bars represent SEM. *, P < 0.05 compared with vector alone.

Mentions: Because focal complex formation is critical to cell migration, we next asked whether TRAF4–Hic-5 interactions were important for such migration. First, we found that short inhibitory RNA (siRNA)–mediated knockdown of either endogenous TRAF4 or Hic-5 diminished HUVEC migration across fibronectin-coated filters in response to a VEGF gradient (Fig. 4 A). Furthermore, the superoxide dismutase mimetic MnTMPyP also inhibited endothelial cell migration, which is consistent with a role for oxidants in migration. Next, to determine relevant binding domains, truncations of each protein were expressed in vivo to preserve zinc finger structures. COOH-terminal truncations of Hic-5, which resulted in serial loss of the four COOH-terminal LIM domains and three LD motifs, revealed that only full-length Hic-5 coprecipitated with TRAF4 (Fig. 4 B), indicating that the COOH-terminal LIM domain 4 (residues 388–444) was necessary for TRAF4 binding. In addition, the Hic-5 LIM domain 4 in isolation did not coprecipitate with TRAF4, whereas the tandem LIM domains 3 and 4 (L3,4) did (Fig. 4 C). Thus, LIM domains 3 and 4 are each necessary but insufficient for TRAF4 binding. Conversely, full-length Hic-5 coprecipitated the COOH-terminal TRAF domain of TRAF4 (TRAF4-C) but not the NH2-terminal remainder containing the ring and zinc fingers (Fig. 4 D). Thus Hic-5(L3,4) binds the COOH terminus of TRAF4.


Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

TRAF4–Hic-5 interactions affect endothelial cell migration. (A) HUVEC were transfected with siRNA against either TRAF4 or Hic-5, and migration across filters in response to a VEGF gradient was measured. Immunoblots show protein levels 48 h after transfection. Control siRNA was against luciferase. 100 μM MnTMPyP was present only during the 16-h migration period. Knockdown of Hic-5 or TRAF4 or antioxidant treatment decreased VEGF-induced migration of HUVEC. hpf, high-powered field. (B) Phoenix-293 cells were cotransfected with full-length TRAF4 and Flag-tagged Hic-5 truncations as indicated. Immunoblots of lysates before TRAF4 immunoprecipitation are shown as input. Cartoon below diagrams Hic-5 truncations showing NH2-terminal Flag tag, three LD motifs (shaded), and four COOH-terminal LIM domains (open). Only full-length Flag–Hic-5 containing LIM 4 coprecipitated with TRAF4 (first lane). (C) Phoenix-293 cells were cotransfected with full-length TRAF4 and full-length Flag–Hic-5 (FL), Flag–Hic-5(329–444) (LIM3,4), or Flag–Hic-5(388–444) (LIM4). Lysates before TRAF4 immunoprecipitation were immunoblotted for Hic-5 and reprobed for TRAF4 to show input. Top panel shows coprecipitation of full-length Flag–Hic-5 and Flag–Hic-5(329–444) but not Flag–Hic-5(388–444) with TRAF4. (D) Phoenix-293 cells were cotransfected with full-length Flag–Hic-5 and either Flag-TRAF4(1–260) (TRAF4-N) or Flag-TRAF4(261–470) (TRAF4-C). Lysate was immunoblotted with anti-Flag (bottom) to show Hic-5 and TRAF4 inputs simultaneously. After immunoprecipitation with anti–Hic-5, blots were probed with anti-Flag to demonstrate the recovery of Flag–TRAF4-C but not Flag–TRAF4-N. Ig heavy and light chains (HC and LC) are shown. (E) HUVEC were cotransfected with pEGFP and empty vector (pCIN), TRAF4-C, or Hic-5(LIM3,4). GFP-expressing cells migrating across 8-μm pore filters in response to a VEGF gradient is shown. Migration in response to VEGF was blocked by the coexpression of either TRAF4-C or Hic-5(LIM3,4). (A and E) *, P < 0.05 compared with no VEGF control; †, P < 0.05 compared with VEGF control. (F) Discrete DsRed-p47 dotlike structures characteristically appeared at the edges of lamellar protrusions (arrows). Bar, 20 μm. Cotransfection of HUVEC with TRAF4-C or knockdown of endogenous TRAF4 or Hic-5 decreased the number of cells (*, P < 0.05) with such DsRed-p47 structures. (G) HUVEC were cotransfected as in D and plated on fibronectin-coated etched coverslips. Phase-contrast and epifluorescent (green) images were obtained immediately after wounding and at 16 and 24 h after wounding. (H) Histogram represents mean speed of GFP-expressing cells migrating into the wound. Entry of GFP-expressing cells into the wound was decreased by the coexpression of TRAF4-C or Hic-5(LIM3,4). Non–GFP-expressing cells migrated into wounds at comparable speeds. Error bars represent SEM. *, P < 0.05 compared with vector alone.
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fig4: TRAF4–Hic-5 interactions affect endothelial cell migration. (A) HUVEC were transfected with siRNA against either TRAF4 or Hic-5, and migration across filters in response to a VEGF gradient was measured. Immunoblots show protein levels 48 h after transfection. Control siRNA was against luciferase. 100 μM MnTMPyP was present only during the 16-h migration period. Knockdown of Hic-5 or TRAF4 or antioxidant treatment decreased VEGF-induced migration of HUVEC. hpf, high-powered field. (B) Phoenix-293 cells were cotransfected with full-length TRAF4 and Flag-tagged Hic-5 truncations as indicated. Immunoblots of lysates before TRAF4 immunoprecipitation are shown as input. Cartoon below diagrams Hic-5 truncations showing NH2-terminal Flag tag, three LD motifs (shaded), and four COOH-terminal LIM domains (open). Only full-length Flag–Hic-5 containing LIM 4 coprecipitated with TRAF4 (first lane). (C) Phoenix-293 cells were cotransfected with full-length TRAF4 and full-length Flag–Hic-5 (FL), Flag–Hic-5(329–444) (LIM3,4), or Flag–Hic-5(388–444) (LIM4). Lysates before TRAF4 immunoprecipitation were immunoblotted for Hic-5 and reprobed for TRAF4 to show input. Top panel shows coprecipitation of full-length Flag–Hic-5 and Flag–Hic-5(329–444) but not Flag–Hic-5(388–444) with TRAF4. (D) Phoenix-293 cells were cotransfected with full-length Flag–Hic-5 and either Flag-TRAF4(1–260) (TRAF4-N) or Flag-TRAF4(261–470) (TRAF4-C). Lysate was immunoblotted with anti-Flag (bottom) to show Hic-5 and TRAF4 inputs simultaneously. After immunoprecipitation with anti–Hic-5, blots were probed with anti-Flag to demonstrate the recovery of Flag–TRAF4-C but not Flag–TRAF4-N. Ig heavy and light chains (HC and LC) are shown. (E) HUVEC were cotransfected with pEGFP and empty vector (pCIN), TRAF4-C, or Hic-5(LIM3,4). GFP-expressing cells migrating across 8-μm pore filters in response to a VEGF gradient is shown. Migration in response to VEGF was blocked by the coexpression of either TRAF4-C or Hic-5(LIM3,4). (A and E) *, P < 0.05 compared with no VEGF control; †, P < 0.05 compared with VEGF control. (F) Discrete DsRed-p47 dotlike structures characteristically appeared at the edges of lamellar protrusions (arrows). Bar, 20 μm. Cotransfection of HUVEC with TRAF4-C or knockdown of endogenous TRAF4 or Hic-5 decreased the number of cells (*, P < 0.05) with such DsRed-p47 structures. (G) HUVEC were cotransfected as in D and plated on fibronectin-coated etched coverslips. Phase-contrast and epifluorescent (green) images were obtained immediately after wounding and at 16 and 24 h after wounding. (H) Histogram represents mean speed of GFP-expressing cells migrating into the wound. Entry of GFP-expressing cells into the wound was decreased by the coexpression of TRAF4-C or Hic-5(LIM3,4). Non–GFP-expressing cells migrated into wounds at comparable speeds. Error bars represent SEM. *, P < 0.05 compared with vector alone.
Mentions: Because focal complex formation is critical to cell migration, we next asked whether TRAF4–Hic-5 interactions were important for such migration. First, we found that short inhibitory RNA (siRNA)–mediated knockdown of either endogenous TRAF4 or Hic-5 diminished HUVEC migration across fibronectin-coated filters in response to a VEGF gradient (Fig. 4 A). Furthermore, the superoxide dismutase mimetic MnTMPyP also inhibited endothelial cell migration, which is consistent with a role for oxidants in migration. Next, to determine relevant binding domains, truncations of each protein were expressed in vivo to preserve zinc finger structures. COOH-terminal truncations of Hic-5, which resulted in serial loss of the four COOH-terminal LIM domains and three LD motifs, revealed that only full-length Hic-5 coprecipitated with TRAF4 (Fig. 4 B), indicating that the COOH-terminal LIM domain 4 (residues 388–444) was necessary for TRAF4 binding. In addition, the Hic-5 LIM domain 4 in isolation did not coprecipitate with TRAF4, whereas the tandem LIM domains 3 and 4 (L3,4) did (Fig. 4 C). Thus, LIM domains 3 and 4 are each necessary but insufficient for TRAF4 binding. Conversely, full-length Hic-5 coprecipitated the COOH-terminal TRAF domain of TRAF4 (TRAF4-C) but not the NH2-terminal remainder containing the ring and zinc fingers (Fig. 4 D). Thus Hic-5(L3,4) binds the COOH terminus of TRAF4.

Bottom Line: Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects.We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

ABSTRACT
Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47(phox) and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

Show MeSH
Related in: MedlinePlus