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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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The IL-3R β common subunit associates with the active form of β1 integrin. (A) Starved MO7-E cells incubated or not with 10 mM of bivalent Mn2+ cations (Mn) with (+) or without (−) IL-3 were detergent extracted. Cell extracts were IP with β1 integrin mAb or IL-3R β common antibodies and IB with IL-3R β common antibodies. (B) MO7-E cells untreated (a–c) or treated with 10 mM Mn2+ (d) were subjected to flow cytometry analysis with IL-1β mAb (a) or preimmune IgG (a–e, left curve) as negative controls, mAb BV7 (b) or mAb 12G10 (c and d). Endothelial cells were subjected to flow cytometry analysis with mAb 12G10 (e). The data expressed as relative cell number (y axis) plotted as a function of fluorescence intensity (x axis) are representative of three experiments. (C) Extracts from MO7-E cells left untreated (−) or treated with Mn2+ (+) were subjected to SDS-PAGE and IB with phospho-STAT5 antibody and STAT5A antibody. Similar results were obtained in four individual experiments.
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fig3: The IL-3R β common subunit associates with the active form of β1 integrin. (A) Starved MO7-E cells incubated or not with 10 mM of bivalent Mn2+ cations (Mn) with (+) or without (−) IL-3 were detergent extracted. Cell extracts were IP with β1 integrin mAb or IL-3R β common antibodies and IB with IL-3R β common antibodies. (B) MO7-E cells untreated (a–c) or treated with 10 mM Mn2+ (d) were subjected to flow cytometry analysis with IL-1β mAb (a) or preimmune IgG (a–e, left curve) as negative controls, mAb BV7 (b) or mAb 12G10 (c and d). Endothelial cells were subjected to flow cytometry analysis with mAb 12G10 (e). The data expressed as relative cell number (y axis) plotted as a function of fluorescence intensity (x axis) are representative of three experiments. (C) Extracts from MO7-E cells left untreated (−) or treated with Mn2+ (+) were subjected to SDS-PAGE and IB with phospho-STAT5 antibody and STAT5A antibody. Similar results were obtained in four individual experiments.

Mentions: To investigate further the molecular mechanisms leading to adhesion-independent constitutive association of IL-3R β common with β1 integrin, the hematopoietic MO7-E cells, which express both IL-3R (Brizzi et al., 1994) and β1 integrin, was used. Consistent with the fact that in these cells β1 integrin is present as an inactive form that can be activated by bivalent cations (Bazzoni et al., 1995; Mould et al., 1995; Humphries et al., 2003), we found that the β1 integrin was able to coimmunoprecipitate the IL-3R β common only in cells treated with Mn2+ (Fig. 3 A). These data suggest that activation of β1 integrin is a prerequisite for association with the IL-3R β common. Flow cytometry analysis using mAb 12G10, recognizing one epitope expressed only in the FN-competent β1 integrin (Mould et al., 1995), or mAb BV7, recognizing a constitutive β1 integrin epitope, was performed on MO7-E cells and endothelial cells. As shown in Fig. 3 B, mAb 12G10 binds to MO7-E cells only in the presence of the activating bivalent cation Mn2+ (Fig. 3 B, compare c and d). In contrast, in endothelial cells the 12G10-recognizing epitope was already exposed in the absence of any treatment (Fig. 3 B, e), indicating that a large majority of the β1 integrin expressed on the surface of endothelial cells was in the active state. No staining was observed by incubating cells with anti–IL-1β (Fig. 3 B, a) as well as with preimmune IgG (Fig. 3 B, a–e, left curve). Thus, IL-3R β common interacts with β1 integrin only in its active form, indicating that β1 integrin activation allows its association with the IL-3R.


{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)

The IL-3R β common subunit associates with the active form of β1 integrin. (A) Starved MO7-E cells incubated or not with 10 mM of bivalent Mn2+ cations (Mn) with (+) or without (−) IL-3 were detergent extracted. Cell extracts were IP with β1 integrin mAb or IL-3R β common antibodies and IB with IL-3R β common antibodies. (B) MO7-E cells untreated (a–c) or treated with 10 mM Mn2+ (d) were subjected to flow cytometry analysis with IL-1β mAb (a) or preimmune IgG (a–e, left curve) as negative controls, mAb BV7 (b) or mAb 12G10 (c and d). Endothelial cells were subjected to flow cytometry analysis with mAb 12G10 (e). The data expressed as relative cell number (y axis) plotted as a function of fluorescence intensity (x axis) are representative of three experiments. (C) Extracts from MO7-E cells left untreated (−) or treated with Mn2+ (+) were subjected to SDS-PAGE and IB with phospho-STAT5 antibody and STAT5A antibody. Similar results were obtained in four individual experiments.
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fig3: The IL-3R β common subunit associates with the active form of β1 integrin. (A) Starved MO7-E cells incubated or not with 10 mM of bivalent Mn2+ cations (Mn) with (+) or without (−) IL-3 were detergent extracted. Cell extracts were IP with β1 integrin mAb or IL-3R β common antibodies and IB with IL-3R β common antibodies. (B) MO7-E cells untreated (a–c) or treated with 10 mM Mn2+ (d) were subjected to flow cytometry analysis with IL-1β mAb (a) or preimmune IgG (a–e, left curve) as negative controls, mAb BV7 (b) or mAb 12G10 (c and d). Endothelial cells were subjected to flow cytometry analysis with mAb 12G10 (e). The data expressed as relative cell number (y axis) plotted as a function of fluorescence intensity (x axis) are representative of three experiments. (C) Extracts from MO7-E cells left untreated (−) or treated with Mn2+ (+) were subjected to SDS-PAGE and IB with phospho-STAT5 antibody and STAT5A antibody. Similar results were obtained in four individual experiments.
Mentions: To investigate further the molecular mechanisms leading to adhesion-independent constitutive association of IL-3R β common with β1 integrin, the hematopoietic MO7-E cells, which express both IL-3R (Brizzi et al., 1994) and β1 integrin, was used. Consistent with the fact that in these cells β1 integrin is present as an inactive form that can be activated by bivalent cations (Bazzoni et al., 1995; Mould et al., 1995; Humphries et al., 2003), we found that the β1 integrin was able to coimmunoprecipitate the IL-3R β common only in cells treated with Mn2+ (Fig. 3 A). These data suggest that activation of β1 integrin is a prerequisite for association with the IL-3R β common. Flow cytometry analysis using mAb 12G10, recognizing one epitope expressed only in the FN-competent β1 integrin (Mould et al., 1995), or mAb BV7, recognizing a constitutive β1 integrin epitope, was performed on MO7-E cells and endothelial cells. As shown in Fig. 3 B, mAb 12G10 binds to MO7-E cells only in the presence of the activating bivalent cation Mn2+ (Fig. 3 B, compare c and d). In contrast, in endothelial cells the 12G10-recognizing epitope was already exposed in the absence of any treatment (Fig. 3 B, e), indicating that a large majority of the β1 integrin expressed on the surface of endothelial cells was in the active state. No staining was observed by incubating cells with anti–IL-1β (Fig. 3 B, a) as well as with preimmune IgG (Fig. 3 B, a–e, left curve). Thus, IL-3R β common interacts with β1 integrin only in its active form, indicating that β1 integrin activation allows its association with the IL-3R.

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