QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 2 October 2007
doi: 10.1242/jcs.03484

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Friedland, J. C. Articles by Weaver, V. M. Search for Related Content PubMed PubMed Citation Articles by Friedland, J. C. Articles by Weaver, V. M. Social Bookmarking                         What's this?

64 integrin activates Rac-dependent p21-activated kinase 1 to drive NF-B-dependent resistance to apoptosis in 3D mammary acini

Julie C. Friedland^1,2,3,*, Johnathon N. Lakins^2,3,4,5, Marcelo G. Kazanietz¹, Jonathan Chernoff⁶, David Boettiger⁷ and Valerie M. Weaver^2,3,4,5,

¹ Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA
² Department of Surgery, University of California San Francisco, San Francisco, CA 94143, USA
³ Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA
⁴ Department of Pathology, University of Pennsylvania, Philadelphia, PA 19104, USA
⁵ Center for Bioengineering and Tissue Regeneration, University of California San Franisco, San Francisco, CA 94143, USA
⁶ Fox Chase Cancer Center, Philadelphia, PA 19111, USA
⁷ Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104, USA

View larger version (16K): [in this window] [in a new window]   Fig. 1. Tissue differentiation is associated with an increase in (6)4 integrin-dependent Rac activity and enhanced resistance to apoptosis. (A) Dose-response curves showing that nonmalignant HMT-3522 S1 and MCF10A MECs acquire resistance to apoptosis induced by chemotherapeutics including taxol and immune receptor activators such as Trail following their rBM-induced differentiation into 3D polarized acini. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive cells 48 hours after treatment divided by the total cell number. MECs were either plated on top of a 1:100 diluted rBM for 48-96 hours (2D) or differentiated by embedment within rBM for 10-12 days (3D) followed by exposure to increasing doses of apoptotic stimuli. (B) Representative immunoblot of immunoprecipitated Pak-associated Rac (GTP-Rac), total Rac (Rac) and E-cadherin in MECs plated either on top (2D) as monolayers or within (3D) rBM to assemble acini. The data indicate that total Rac decreases noticeably following rBM-induced differentiation, whereas GTP-loaded Rac increases dramatically. (C) Average relative specific activity of Rac in 3D mammary acini calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization, as shown in B. (D) Representative immunoblot of GTP-Rac, total Rac (Rac) and E-cadherin in vector control and tail-less 4 integrin (4cyto) 3D mammary acini grown within rBM (10-14 days). The data illustrate that Rac activity diminishes significantly in mammary acini that express the signaling-defective tail-less 4 integrin. (E) Average relative specific activity of Rac in control mammary acini versus mammary tissues expressing the tail-less 4 integrin calculated as above in C and shown in D. Results are the mean±s.e.m. of three to five separate experiments. *P0.05; **P0.01; ***P0.001.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Rac activity is necessary for resistance to apoptosis in 3D mammary acini. (A) FACS analysis showing increased EGFP expression in MECs expressing the EGFP-tagged N17 Rac (P4) in comparison with vector control MECs (P3). (B) Representative immunoblot of GTP-Rac, Rac and E-cadherin in vector control MECs grown as 2D monolayers in comparison with MECs expressing EGFP-tagged N17 Rac. The data illustrate that N17Rac significantly reduces GTP-Rac levels in MECs. (C) Average relative specific activity of Rac in MECs calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization of data illustrated in B. (D) Representative immunoblot of phospho-Pak1 and total Pak1 in 2D monolayer cultures of control MECs and MECs expressing EGFP-tagged N17Rac demonstrating how loss of Rac activity also reduces Pak1 activity. (E) Bar graph depicting the average degree of reduction of Pak1 activity in MECs expressing EGFP-N17Rac in comparison with control MECs. (F) Dose-response curves of the percentage apoptosis, as determined by calculating the number of activated caspase-3-positive cells divided by the total cell number, showing how 3D rBM polarized mammary acini with reduced Rac activity are now more sensitive to both chemotherapeutic (taxol) and receptor-mediated (Trail) apoptotic stimuli. Results are the mean±s.e.m. of three to five separate experiments. *P0.05 (C,E,F); **P0.01 (F).

View larger version (29K): [in this window] [in a new window]   Fig. 3. Rac-mediates resistance to apoptosis and changes in tissue polarity through distinct mechanisms. (A) (top) Phase-contrast and confocal immunofluorescence images of colonies of control mammary acini (left panel; control), acini expressing EGFP-tagged N17Rac (middle panel; N17Rac) and acini treated with the Rac1 inhibitor NSC23766 (right panel; NSC23766) stained for 4 integrin, laminin-332, collagen IV and scribble (red). The data show that expression of N17Rac affects neither tissue integrity (compare regions highlighted by arrows in control phase-contrast images of acini with regions highlighted by arrows in images of N17Rac-expressing acini; top) nor tissue polarity (lower images; evidenced by similar basally localized 4 integrin; deposition of laminin-332 and collagen IV and intact cell-cell-localized scribble; compare images of control acini with images of acini expressing N17Rac). However, treatment of acini with the Rac inhibitor NSC23766 disrupted the integrity of acini (compare regions of image highlighted by arrows in phase-contrast images of control with images of NSC23766-treated acini) and severely perturbed tissue polarity, as evidenced by disturbed localization of 4 integrin, laminin-332, collagen IV and scribble (compare images of control with images of NSC23766-treated acini). Bar, 50 µm. (B) Representative immunoblot of GTP-Rac, Rac and E-cadherin in vector control MECs grown as 3D acini in comparison to MECs expressing EGFP-tagged N17 Rac and control acini treated with the specific Rac inhibitor NSC23766. The data illustrate that while N17Rac partially reduces GTP-Rac levels in 3D mammary acini, treatment with the Rac inhibitor decreases Rac activity to barely detectable levels. (C) Average relative specific activity of Rac in MECs calculated by densitometric analysis of immunoblots of GTP-Rac divided by total cellular Rac following E-cadherin normalization of data illustrated in B. (D) Bar graphs illustrating increased percentage of apoptotic cells induced by treatment with Trail (1 µg/ml) (left) or Taxol (40 µM) right in 3D rBM differentiated MECs with significantly reduced Rac activity mediated by treatment with the specific Rac inhibitor NSC23766. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive MECs divided by the total number of MECs 24 hours following treatment with Trail or Taxol. Results are the mean ± s.e.m. of three to five separate experiments. *P0.05; **P0.01.

View larger version (24K): [in this window] [in a new window]   Fig. 4. GAP-dependent Rac inactivation sensitizes mammary acini to induction of death. (A) Representative immunoblot showing non-detectable amounts of hemagglutinin (HA) expressed in vector control MECs and high levels in MECs expressing an exogenous HA-tagged 2-chimerin. (B) Representative immunoblot showing GTP-Rac, Rac and E-cadherin levels in vector control MECs in comparison with 2-chimerin-expressing MECs. (C) Average relative specific-activity of GTP-loaded Rac illustrating reduced Rac activity in MEC-expressing exogenous 2-chimerin in comparison with vector control MECs. (D) Bar graph illustrating increased percentage of apoptotic cells induced by treatment with Trail (1 µg/ml) in 3D rBM differentiated MECs with reduced Rac activity mediated by expression of either the Rac GAP 2-chimerin or dominant-negative N17Rac. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive MECs divided by the total number of MECs 24 hours following treatment with Trail. Similar results were obtained following treatment with chemotherapeutic agents (data not shown). (E) (top) Phase-contrast and (bottom) immunofluorescence images of 3D poly-HEMA rBM-generated vector control MEC colonies (left) and MEC colonies expressing HA-tagged 2-chimerin (right; red) illustrating robust and uniform expression of the transgene 48 hours following adenoviral infection. Bar, 50 µm. Arrows indicate the representative phenotype of acini morphology. Bar, 50 µm. Results are the mean ± s.e.m. of three separate experiments. *P0.05; **P0.01.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Rac-dependent Pak1 activity is necessary and sufficient for the resistance to apoptosis of mammary acini. (A) Representative immunoblots of phospho-Pak1, total Pak1, Pak-3, phospho-Pak4, total Pak4 and E-cadherin in rBM-ligated MECs plated as 2D monolayers or 3D mammary acini. The data indicate that Pak1 activity is significantly higher, although the abundance of Pak4 is higher in 3D the specific activity of Pak4 is substantially lower and Pak3 is non-detectable in 3D mammary acini in comparison with 2D monolayers of MECs. (B) Quantification of averaged experimental data shown in A of Pak1 specific activity, calculated by densitometric analysis of immunoblots of phospho-Pak1 divided by total Pak1 after normalization to E-cadherin. Similar results were obtained for HMT-3522 S1 and MCF10A nonmalignant MECs. (C) Representative immunoblots of phospho-Pak1 and total Pak1 in 3D mammary acini expressing the tail-less 4 integrin (4cyto) in comparison with control acini. The data demonstrate that Pak1 activity, but not expression, is regulated by (6)4 integrin signaling. (D) Quantification of averaged experimental data shown in C of Pak1 specific activity calculated as described above in B. (E) Dose-response curves illustrating the percentage apoptosis induced in 3D mammary acini following 24 hours of treatment with increasing concentrations of Trail (left) and taxol (right), calculated by scoring the number of caspase-3-positive cells divided by the total number of cells. Mammary acini with reduced Rac activity were sensitized to Trail and taxol-induced apoptosis (N17Rac vector) but their death-resistance phenotype was restored following ectopic expression of V12Rac. (F) Bar graphs illustrating how expression of V12Rac restores resistance to apoptosis induced by Trail treatment in 3D mammary acini expressing N17Rac, whereas expression of V12Rac H40, which cannot activate Pak, does not restore resistance. The percentage apoptosis was calculated by scoring the number of activated caspase-3-positive cells divided by the total number of cells. (G) Bar graph demonstrating that inhibiting Pak activity, by expressing PID significantly sensitizes mammary acini to Trail-induced death, analogous to that mediated by N17Rac. (H) Bar graph showing how expression of wild-type Pak1 can restore resistance to Trail treatment to 3D mammary acini expressing N17Rac. Results are the mean ± s.e.m. of three to five separate experiments. *P0.05; **P0.01.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Pak permits activation of NF-B to mediate resistance to apoptosis in mammary acini. (A) Bar graph indicating how inhibiting activation of NF-B by treating mammary acini with SN50 peptide permits Trail-induced apoptosis. MECs were grown in rBM for ten days and treated with SN50 or inactive, scrambled SN50M peptide. Polarized acini were treated with Trail (1 µg/ml) and after 24 hours the acini were stained and quantified for activated caspase 3. (B) Bar graph illustrating how ectopically expressed wild-type Pak1 can restore resistance to apoptosis to N17Rac-expressing mammary acini treated with Trail (N17Rac), but the acini remain death sensitive if NF-B activation is prevented by pre-incubation with SN50. 3D mammary acini were infected with adenovirus, pre-incubated for 24 hours with either SN50 to inhibit NF-B activation or its non-active analogue SN50M, and treated with Trail (1 µg/ml) for 24 hours. The percentage apoptosis for (A) and (B) was calculated by scoring the number of activated caspase-3-positive cells divided by the total cell number. (C) Confocal immunofluorescence microscopy images showing NF-B p65 nuclear translocation in response to treatment with Trail (90 min) in 3D mammary acini expressing either vector (control), the Pak activity inhibitor (PID), N17Rac (N17Rac) or N17Rac and wild-type Pak1 (N17Rac/Pak1 WT). Note the presence of high nuclear levels of p65, as indicated by "n" and identified by arrow, in response to Trail stimulation in control and N17Rac/Pak1-WT-expressing mammary tissues and decreased levels in acini with reduced Rac or Pak activity. Bar, 10 µm. n, nucleus. (D) Quantification of nuclear p65 in 50-100 representative images as shown in C. (E) Bar graph showing how expression of a wild-type p65 transgene restores Trail-induced death-resistance to apoptosis-sensitized N17Rac- and PID-expressing mammary acini. (F) Bar graph showing how inhibiting activation of NF-B through expression of the IBM super-repressor permits Trail-dependent induction of death in 3D mammary acini despite elevated levels of Pak1. Results are the mean ± s.e.m. of three separate experiments. *P0.05; **P0.01; ***P0.001.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Schematic illustrating proposed mechanism whereby 64 integrin regulates Rac and Pak to mediate Bad- and NF-B-dependent resistance to apoptosis in mammary acini. Nonmalignant MECs differentiate into polarized-tissue-like structures that assemble an endogenous laminin-332-containing basement membrane. Laminin-ligation of 64 integrin stimulates Rac to drive tissue polarity and promote resistance to apoptosis in 3D mammary acini through Pak-dependent activation of NF-B and phosphorylation of Bad. LM-332, laminin-5; P, phosphorylated.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter