TGFbeta1 signaling via alphaVbeta6 integrin

Abstract

Background: Transforming growth factor beta1 (TGFbeta1) is a potent inhibitor of epithelial cell growth, thus playing an important role in tissue homeostasis. Most carcinoma cells exhibit a reduced sensitivity for TGFbeta1 mediated growth inhibition, suggesting TGFbeta1 participation in the development of these cancers. The tumor suppressor gene DPC4/SMAD4, which is frequently inactivated in carcinoma cells, has been described as a key player in TGFbeta1 mediated growth inhibition. However, some carcinoma cells lacking functional SMAD4 are sensitive to TGFbeta1 induced growth inhibition, thus requiring a SMAD4 independent TGFbeta1 pathway.

Results: Here we report that mature TGFbeta1 is a ligand for the integrin alphaVbeta6, independent of the common integrin binding sequence motif RGD. After TGFbeta1 binds to alphaVbeta6 integrin, different signaling proteins are activated in TGFbeta1-sensitive carcinoma cells, but not in cells that are insensitive to TGFbeta1. Among others, interaction of TGFbeta1 with the alphaVbeta6 integrin resulted in an upregulation of the cell cycle inhibitors p21/WAF1 and p27 leading to growth inhibition in SMAD4 deleted as well as in SMAD4 wildtype carcinoma cells.

Conclusions: Our data provide support for the existence of an alternate TGFbeta1 signaling pathway that is independent of the known SMAD pathway. This alternate pathway involves alphaVbeta6 integrin and the Ras/MAP kinase pathway and does not employ an RGD motif in TGFbeta1-sensitive tumor cells. The combined action of these two pathways seems to be necessary to elicit a complete TGFbeta1 signal.

Figure 1

Colocalization of TGFβ₁, α_Vβ₆ integrin and the cytoskeleton. Panc-1 cells were stimulated with mature TGFβ₁ and stained using anti TGFβ₁ (labeled with goat anti-rabbit IgG conjugate, A-11046), α_V/β₆ (labeled with goat anti-rabbit IgG conjugate, A-11046) and Actin antibodies. Magnification 1000×.

Figure 2

Phosphorylation and immobilization of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored α_Vβ₆ was immunoprecipitated after TGFβ₁ stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A) or Western blotting after biotinylation of all proteins and streptavidin detection (B). Presence of TGFβ₁ (C), α_V and β₆ integrin (D) in the co-precipitates is also demonstrated. TGFβ-receptor-I and II (TGFβRI and TGFβRII) are expressed at nearly equal levels in all cell lines as demonstrated by western blotting from whole cell extracts (E). In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min) or with a TGFβ antibody (15 μg/ml for 30 min).

Figure 3

Enhanced Tyrosine Phosphorylation of proteins associated with the integrin-cytoskeleton-complex. Cytoskeletally anchored α_Vβ₆ was immunoprecipitated after TGFβ₁ and/or fibronectin stimulation (10 nM for 10 minutes) followed by Western analysis with antibodies against tyrosine-phosphorylated proteins (A). Reprobing with α_V and β₆ antibodies show equal anounts of precipitates used (B).

Figure 4

p125^FAK activation by mature TGFβ₁ via integrin α_Vβ₆. Stimulation of BxPC-3 with mature TGFβ₁ (10 nM for 10 minutes), immunoprecipitation with α_V- and β₆ integrin antibodies after preparation of the cytoskeleton, followed by probing with pp125^Fak and p125^FAK antibodies. In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min), with a TGFβ antibody (15 μg/ml for 30 min), cytochalasin D and BAPTA AM, respectively.

Figure 5

Cell cycle genes in response to TGFβ₁. Western Blot analysis of HeLa cells stimulated with 10 nM of mature TGFβ₁ for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively.

Figure 6

Enhanced level of cytoskeletal anchored proteins in response to TGFβ₁ (A). Western Blot analysis of BxPC-3 and HeLa cells as indicated after stimulation with TGFβ₁ for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively. Purity of the TGFβ₁ used (B). Ten nanogram of mature TGFβ₁ and latent TGFβ₁ were subjected to non-reducing SDS-PAGE dollowed by silver staining. No latant TGFβ₁ could be detected in the mature TGFβ₁ used for stimulation. BxPC-3 cells are SMAD4^-/-(C). One hundred microgram of whole cell extract from BxPC-3 and HeLa cells were probed with p125^FAK and SMAD4 antibodies on the same membrane. As reported, BxPC-3 cells are found to be SMAD4^-/-.

Figure 7

Cell cycle genes in response to TGFβ₁. Western Blot analysis of MCF-7 and MDA-MB 231 cells as indicated after stimulation with TGFβ₁ for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively.

Figure 8

Cell cycle genes in response to TGFβ₁. Western Blot analysis of BxPC-3, LOVO, SW48 and DLD1 cells as indicated after stimulation with TGFβ₁ for the time indicated. Cytoskeletally anchored proteins are differentially marked. In part the cells were preincubated with α_V- and β₆-antibodies (1:100 each for 30 min), with a TGFβ-RII antibody (15 μg/ml for 30 min), cytochalasin D, BAPTA AM and MEK1 inhibitor PD98059, respectively.

Figure 9

Activation and nuclear translocation of SMAD2/3 in response to TGFβ₁ (A). Nuclear and cytoplasmatic fraction of cellular proteins (BxPC-3) after stimulation with 10 nM of TGFβ₁ for 10 minutes and Western blot analysis for SMAD2/3 and phosphorylated SMAD2/3. Purity of cytoplasmic and nuclear fraction (B). Cytoplasmic and nuclear extracts from K562 cells were probed with p125^FAK, PCNA and Iκ Bα antibodies at the same time. As predicted, p125^FAK cold exclusively be detected in the cytoplasmic extract, whereas PCNA is found in the nucleus. Iκ Bα served as loading control.

Figure 10

Hypothesis about an alternate TGFβ₁ signaling pathway via α_Vβ₆ integrin, independent of RGD. This pathway may be required for full TGFβ₁ induced transcriptional activation, which explains the TGFβ₁ sensitivity of those cells lacking DPC4/SMAD4 function that still react with growth inhibition.