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{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation.

Defilippi P, Rosso A, Dentelli P, Calvi C, Garbarino G, Tarone G, Pegoraro L, Brizzi MF - J. Cell Biol. (2005)

Bottom Line: Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry.Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression.Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126, Torino, Italy. paola.defilippi@unito.it

ABSTRACT
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

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Related in: MedlinePlus

FN-induced association of activated STAT5A with the constitutive β1 integrin–IL-3R β common complex. (A) Cell extracts from starved endothelial cells kept in suspension (S) or plated on FN-coated dishes (20 min) were immunoprecipitated (IP) with mAb TS2/16 to the β1 integrin subunit, and the immunoprecipitates were divided in two aliquots. Western blot was performed with IL-3R β common antibodies (top left) or with the same antibodies preadsorbed on the recombinant IL-3R β common protein (bottom left). In the absence of the positive signal, to reveal IgG, the latter filter was overexposed. The same amount of cell extracts was IP with IL-3R β common antibodies, the immunoprecipitate run on a 6% SDS-PAGE in non-reducing conditions, and IB with β1 integrin mAb (top right). Parallel experiments were IP with anti-β2 microglobulin antibodies. Filters were IB with IL-3R β common antibodies (bottom right). (B) Cell extracts from endothelial cells plated on FN-coated dishes for different times in the presence of 50 μM pervanadate were IP with IL-3R β common antibodies and IB with antiphosphotyrosine antibodies (α PY; top) and reprobed with IL-3R β common antibodies (bottom). (C) Cell extracts from endothelial cells plated as in A were IP with IL-3R β common antibodies, divided in two aliquots, and IB with anti–phospho-STAT5, anti-STAT5A protein, and reimmunoblotted with IL-3R β common antibody or β1 integrin mAb. (D) Cells extracts treated as in A were IP with uncoupled or with STAT5A-coupled Sepharose protein A beads and IB with STAT5A antibody. M07-E cell extracts were used as positive control (+). The results are representative of four independent experiments.
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fig2: FN-induced association of activated STAT5A with the constitutive β1 integrin–IL-3R β common complex. (A) Cell extracts from starved endothelial cells kept in suspension (S) or plated on FN-coated dishes (20 min) were immunoprecipitated (IP) with mAb TS2/16 to the β1 integrin subunit, and the immunoprecipitates were divided in two aliquots. Western blot was performed with IL-3R β common antibodies (top left) or with the same antibodies preadsorbed on the recombinant IL-3R β common protein (bottom left). In the absence of the positive signal, to reveal IgG, the latter filter was overexposed. The same amount of cell extracts was IP with IL-3R β common antibodies, the immunoprecipitate run on a 6% SDS-PAGE in non-reducing conditions, and IB with β1 integrin mAb (top right). Parallel experiments were IP with anti-β2 microglobulin antibodies. Filters were IB with IL-3R β common antibodies (bottom right). (B) Cell extracts from endothelial cells plated on FN-coated dishes for different times in the presence of 50 μM pervanadate were IP with IL-3R β common antibodies and IB with antiphosphotyrosine antibodies (α PY; top) and reprobed with IL-3R β common antibodies (bottom). (C) Cell extracts from endothelial cells plated as in A were IP with IL-3R β common antibodies, divided in two aliquots, and IB with anti–phospho-STAT5, anti-STAT5A protein, and reimmunoblotted with IL-3R β common antibody or β1 integrin mAb. (D) Cells extracts treated as in A were IP with uncoupled or with STAT5A-coupled Sepharose protein A beads and IB with STAT5A antibody. M07-E cell extracts were used as positive control (+). The results are representative of four independent experiments.

Mentions: It is well established that integrins trigger specific signaling pathways by directly trans-activating growth factor receptors (Moro et al., 1998; Miranti and Brugge, 2002; Schwartz and Ginsberg, 2002; Giancotti and Tarone, 2003). We showed that in endothelial cells integrins induce ligand-independent STAT5A activation and c-fos gene expression (Brizzi et al., 1999). As shown in Table I, adhesion of endothelial cells to fibronectin (FN) in the absence of IL-3 is not sufficient to promote entry in the S phase of the cell cycle. Consistently, upon adhesion, unlike c-fos mRNA expression, which is strongly up-regulated (Fig. 1 A), cyclin D1 protein is not modified within 15 h of adhesion (Fig. 1 B, gray bars). Its expression is induced by serum in adherent cells (Fig. 1 B, black bars), confirming that in primary cells expression of G1 phase cyclins requires concomitant signals emanated from integrins and soluble ligands (Assoian and Schwartz, 2001). Endothelial cells plated on FN undergo both proliferation and migration in response to IL-3 treatment (Table I), suggesting that IL-3 and its receptor play a crucial role in regulating endothelial cell functions. The fact that STAT5 is a well defined target of IL-3R signaling in endothelial cells (Dentelli et al., 1999) prompted us to evaluate the involvement of the IL-3R in mediating integrin-dependent STAT5 activation. A likely mechanism through which integrins can activate STAT5A may depend on cross talk between β1 integrin and the IL-3R β common. To investigate this possibility, β1 integrin and IL-3R β common were immunoprecipitated (IP) from endothelial cell extracts and the immunoprecipitates were reciprocally immunoblotted (IB) as shown in Fig. 2 A (top panels). Cell extract from MO7-E cells was IP with an antiserum to IL-3R β common and used as positive control (+). These experiments demonstrate that β1 integrin associates with the IL-3R β common, both in cells kept in suspension or adherent to FN, indicating that these two molecules stably interact in endothelial cells independently from the adhesive state. The specificity of the association was also demonstrated by the loss of signal when the same filter was IB with the IL-3 β common antiserum preadsorbed with the immunizing peptide (Fig. 2 A, bottom left). Moreover, no coimmunoprecipitation of IL-3R β common was observed with a control antibody against β2 microglobulin (Fig. 2 A, bottom right).


{beta}1 Integrin and IL-3R coordinately regulate STAT5 activation and anchorage-dependent proliferation.

Defilippi P, Rosso A, Dentelli P, Calvi C, Garbarino G, Tarone G, Pegoraro L, Brizzi MF - J. Cell Biol. (2005)

FN-induced association of activated STAT5A with the constitutive β1 integrin–IL-3R β common complex. (A) Cell extracts from starved endothelial cells kept in suspension (S) or plated on FN-coated dishes (20 min) were immunoprecipitated (IP) with mAb TS2/16 to the β1 integrin subunit, and the immunoprecipitates were divided in two aliquots. Western blot was performed with IL-3R β common antibodies (top left) or with the same antibodies preadsorbed on the recombinant IL-3R β common protein (bottom left). In the absence of the positive signal, to reveal IgG, the latter filter was overexposed. The same amount of cell extracts was IP with IL-3R β common antibodies, the immunoprecipitate run on a 6% SDS-PAGE in non-reducing conditions, and IB with β1 integrin mAb (top right). Parallel experiments were IP with anti-β2 microglobulin antibodies. Filters were IB with IL-3R β common antibodies (bottom right). (B) Cell extracts from endothelial cells plated on FN-coated dishes for different times in the presence of 50 μM pervanadate were IP with IL-3R β common antibodies and IB with antiphosphotyrosine antibodies (α PY; top) and reprobed with IL-3R β common antibodies (bottom). (C) Cell extracts from endothelial cells plated as in A were IP with IL-3R β common antibodies, divided in two aliquots, and IB with anti–phospho-STAT5, anti-STAT5A protein, and reimmunoblotted with IL-3R β common antibody or β1 integrin mAb. (D) Cells extracts treated as in A were IP with uncoupled or with STAT5A-coupled Sepharose protein A beads and IB with STAT5A antibody. M07-E cell extracts were used as positive control (+). The results are representative of four independent experiments.
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Related In: Results  -  Collection

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fig2: FN-induced association of activated STAT5A with the constitutive β1 integrin–IL-3R β common complex. (A) Cell extracts from starved endothelial cells kept in suspension (S) or plated on FN-coated dishes (20 min) were immunoprecipitated (IP) with mAb TS2/16 to the β1 integrin subunit, and the immunoprecipitates were divided in two aliquots. Western blot was performed with IL-3R β common antibodies (top left) or with the same antibodies preadsorbed on the recombinant IL-3R β common protein (bottom left). In the absence of the positive signal, to reveal IgG, the latter filter was overexposed. The same amount of cell extracts was IP with IL-3R β common antibodies, the immunoprecipitate run on a 6% SDS-PAGE in non-reducing conditions, and IB with β1 integrin mAb (top right). Parallel experiments were IP with anti-β2 microglobulin antibodies. Filters were IB with IL-3R β common antibodies (bottom right). (B) Cell extracts from endothelial cells plated on FN-coated dishes for different times in the presence of 50 μM pervanadate were IP with IL-3R β common antibodies and IB with antiphosphotyrosine antibodies (α PY; top) and reprobed with IL-3R β common antibodies (bottom). (C) Cell extracts from endothelial cells plated as in A were IP with IL-3R β common antibodies, divided in two aliquots, and IB with anti–phospho-STAT5, anti-STAT5A protein, and reimmunoblotted with IL-3R β common antibody or β1 integrin mAb. (D) Cells extracts treated as in A were IP with uncoupled or with STAT5A-coupled Sepharose protein A beads and IB with STAT5A antibody. M07-E cell extracts were used as positive control (+). The results are representative of four independent experiments.
Mentions: It is well established that integrins trigger specific signaling pathways by directly trans-activating growth factor receptors (Moro et al., 1998; Miranti and Brugge, 2002; Schwartz and Ginsberg, 2002; Giancotti and Tarone, 2003). We showed that in endothelial cells integrins induce ligand-independent STAT5A activation and c-fos gene expression (Brizzi et al., 1999). As shown in Table I, adhesion of endothelial cells to fibronectin (FN) in the absence of IL-3 is not sufficient to promote entry in the S phase of the cell cycle. Consistently, upon adhesion, unlike c-fos mRNA expression, which is strongly up-regulated (Fig. 1 A), cyclin D1 protein is not modified within 15 h of adhesion (Fig. 1 B, gray bars). Its expression is induced by serum in adherent cells (Fig. 1 B, black bars), confirming that in primary cells expression of G1 phase cyclins requires concomitant signals emanated from integrins and soluble ligands (Assoian and Schwartz, 2001). Endothelial cells plated on FN undergo both proliferation and migration in response to IL-3 treatment (Table I), suggesting that IL-3 and its receptor play a crucial role in regulating endothelial cell functions. The fact that STAT5 is a well defined target of IL-3R signaling in endothelial cells (Dentelli et al., 1999) prompted us to evaluate the involvement of the IL-3R in mediating integrin-dependent STAT5 activation. A likely mechanism through which integrins can activate STAT5A may depend on cross talk between β1 integrin and the IL-3R β common. To investigate this possibility, β1 integrin and IL-3R β common were immunoprecipitated (IP) from endothelial cell extracts and the immunoprecipitates were reciprocally immunoblotted (IB) as shown in Fig. 2 A (top panels). Cell extract from MO7-E cells was IP with an antiserum to IL-3R β common and used as positive control (+). These experiments demonstrate that β1 integrin associates with the IL-3R β common, both in cells kept in suspension or adherent to FN, indicating that these two molecules stably interact in endothelial cells independently from the adhesive state. The specificity of the association was also demonstrated by the loss of signal when the same filter was IB with the IL-3 β common antiserum preadsorbed with the immunizing peptide (Fig. 2 A, bottom left). Moreover, no coimmunoprecipitation of IL-3R β common was observed with a control antibody against β2 microglobulin (Fig. 2 A, bottom right).

Bottom Line: Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry.Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression.Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Biology and Biochemistry, University of Torino, 10126, Torino, Italy. paola.defilippi@unito.it

ABSTRACT
We previously demonstrated that integrin-dependent adhesion activates STAT5A, a well known target of IL-3-mediated signaling. Here, we show that in endothelial cells the active beta1 integrin constitutively associates with the unphosphorylated IL-3 receptor (IL-3R) beta common subunit. This association is not sufficient for activating downstream signals. Indeed, only upon fibronectin adhesion is Janus Kinase 2 (JAK2) recruited to the beta1 integrin-IL-3R complex and triggers IL-3R beta common phosphorylation, leading to the formation of docking sites for activated STAT5A. These events are IL-3 independent but require the integrity of the IL-3R beta common. IL-3 treatment increases JAK2 activation and STAT5A and STAT5B tyrosine and serine phosphorylation and leads to cell cycle progression in adherent cells. Expression of an inactive STAT5A inhibits cell cycle progression upon IL-3 treatment, identifying integrin-dependent STAT5A activation as a priming event for IL-3-mediated S phase entry. Consistently, overexpression of a constitutive active STAT5A leads to anchorage-independent cell cycle progression. Therefore, these data provide strong evidence that integrin-dependent STAT5A activation controls IL-3-mediated proliferation.

Show MeSH
Related in: MedlinePlus