Limits...
{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.

Show MeSH

Related in: MedlinePlus

IL-3–dependent STAT5 activation requires cell adhesion. (A) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) IL-3 were IB with phospho-STAT5 antibody or STAT5A antibody. Densitometric analysis of phospho-STAT5 is shown in the top panel. The results are the mean ± SD of four independent experiments; *, P < 0.05 control versus experimental groups. (B) Extracts were IP with STAT5A antibodies and IB with phospho-STAT5 antibody or STAT5A antibody. (C) Cell extracts were divided into two aliquots and IP with specific antibodies to STAT5A or STAT5B. The filters were IB with anti-PY and reimmunoblotted with the anti-STAT5A or the anti-STAT5B antibodies. Densitometric analysis of STAT5A phosphorylation, performed as in A, is reported in the top panel. The results are the mean ± SD of four independent experiments done on separate days; *, P < 0.05 control versus experimental groups. (D) Cell extracts were prepared and IP as in B. The filters were IB with a phospho-serine STAT5 antibody (top) that recognizes P-Ser725 on STAT5A or P-Ser730 on STAT5B (Pser) and reimmunoblotted with STAT5A or STAT5B antibodies (bottom). (E) Cell extracts were separately IP with specific antibodies to STAT5A or STAT5B and IB with the anti-PY antibodies (top), STAT5A/STAT5B antibodies (middle), or IL-3R β common antibodies (bottom). The results are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171831&req=5

fig6: IL-3–dependent STAT5 activation requires cell adhesion. (A) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) IL-3 were IB with phospho-STAT5 antibody or STAT5A antibody. Densitometric analysis of phospho-STAT5 is shown in the top panel. The results are the mean ± SD of four independent experiments; *, P < 0.05 control versus experimental groups. (B) Extracts were IP with STAT5A antibodies and IB with phospho-STAT5 antibody or STAT5A antibody. (C) Cell extracts were divided into two aliquots and IP with specific antibodies to STAT5A or STAT5B. The filters were IB with anti-PY and reimmunoblotted with the anti-STAT5A or the anti-STAT5B antibodies. Densitometric analysis of STAT5A phosphorylation, performed as in A, is reported in the top panel. The results are the mean ± SD of four independent experiments done on separate days; *, P < 0.05 control versus experimental groups. (D) Cell extracts were prepared and IP as in B. The filters were IB with a phospho-serine STAT5 antibody (top) that recognizes P-Ser725 on STAT5A or P-Ser730 on STAT5B (Pser) and reimmunoblotted with STAT5A or STAT5B antibodies (bottom). (E) Cell extracts were separately IP with specific antibodies to STAT5A or STAT5B and IB with the anti-PY antibodies (top), STAT5A/STAT5B antibodies (middle), or IL-3R β common antibodies (bottom). The results are representative of four independent experiments.

Mentions: Our experiments, performed in the absence of IL-3, show that in response to adhesion, integrins by activating JAK2 cooperate with the IL-3R β common for the activation of STAT5A. However, when IL-3 was added to endothelial cells plated on FN, not only the level of JAK2 phosphorylation (Fig. 4 E) but also that of STAT5 increased (Fig. 6 A). Kinetics analysis shows that, although FN induced a transient STAT5A phosphorylation, IL-3 addition led to a persistent STAT5A phosphorylation, still detectable within 120 min of treatment (Fig. 6 B). Because it has been previously reported that in endothelial cells, upon IL-3 treatment, either STAT5A or STAT5B undergo activation (Dentelli et al., 1999), endothelial cells kept in suspension or adherent to FN were evaluated for STAT5A and STAT5B activation in response to IL-3. The results presented in Fig. 6 C show that the addition of IL-3 to adherent cells induced an increase in STAT5A phosphorylation, quantified by densitometric analysis (Fig. 6 C, top). Moreover, the amount of STAT5A coimmunoprecipitated with IL-3R β common was also increased in IL-3–treated cells (unpublished data), suggesting that the activation of STAT5A in the IL-3–mediated signaling is the result of a cooperative effect between adhesion and soluble ligand.


{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)

IL-3–dependent STAT5 activation requires cell adhesion. (A) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) IL-3 were IB with phospho-STAT5 antibody or STAT5A antibody. Densitometric analysis of phospho-STAT5 is shown in the top panel. The results are the mean ± SD of four independent experiments; *, P < 0.05 control versus experimental groups. (B) Extracts were IP with STAT5A antibodies and IB with phospho-STAT5 antibody or STAT5A antibody. (C) Cell extracts were divided into two aliquots and IP with specific antibodies to STAT5A or STAT5B. The filters were IB with anti-PY and reimmunoblotted with the anti-STAT5A or the anti-STAT5B antibodies. Densitometric analysis of STAT5A phosphorylation, performed as in A, is reported in the top panel. The results are the mean ± SD of four independent experiments done on separate days; *, P < 0.05 control versus experimental groups. (D) Cell extracts were prepared and IP as in B. The filters were IB with a phospho-serine STAT5 antibody (top) that recognizes P-Ser725 on STAT5A or P-Ser730 on STAT5B (Pser) and reimmunoblotted with STAT5A or STAT5B antibodies (bottom). (E) Cell extracts were separately IP with specific antibodies to STAT5A or STAT5B and IB with the anti-PY antibodies (top), STAT5A/STAT5B antibodies (middle), or IL-3R β common antibodies (bottom). The results are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171831&req=5

fig6: IL-3–dependent STAT5 activation requires cell adhesion. (A) Extracts from endothelial cells kept in suspension or plated on FN-coated dishes with (+) or without (−) IL-3 were IB with phospho-STAT5 antibody or STAT5A antibody. Densitometric analysis of phospho-STAT5 is shown in the top panel. The results are the mean ± SD of four independent experiments; *, P < 0.05 control versus experimental groups. (B) Extracts were IP with STAT5A antibodies and IB with phospho-STAT5 antibody or STAT5A antibody. (C) Cell extracts were divided into two aliquots and IP with specific antibodies to STAT5A or STAT5B. The filters were IB with anti-PY and reimmunoblotted with the anti-STAT5A or the anti-STAT5B antibodies. Densitometric analysis of STAT5A phosphorylation, performed as in A, is reported in the top panel. The results are the mean ± SD of four independent experiments done on separate days; *, P < 0.05 control versus experimental groups. (D) Cell extracts were prepared and IP as in B. The filters were IB with a phospho-serine STAT5 antibody (top) that recognizes P-Ser725 on STAT5A or P-Ser730 on STAT5B (Pser) and reimmunoblotted with STAT5A or STAT5B antibodies (bottom). (E) Cell extracts were separately IP with specific antibodies to STAT5A or STAT5B and IB with the anti-PY antibodies (top), STAT5A/STAT5B antibodies (middle), or IL-3R β common antibodies (bottom). The results are representative of four independent experiments.
Mentions: Our experiments, performed in the absence of IL-3, show that in response to adhesion, integrins by activating JAK2 cooperate with the IL-3R β common for the activation of STAT5A. However, when IL-3 was added to endothelial cells plated on FN, not only the level of JAK2 phosphorylation (Fig. 4 E) but also that of STAT5 increased (Fig. 6 A). Kinetics analysis shows that, although FN induced a transient STAT5A phosphorylation, IL-3 addition led to a persistent STAT5A phosphorylation, still detectable within 120 min of treatment (Fig. 6 B). Because it has been previously reported that in endothelial cells, upon IL-3 treatment, either STAT5A or STAT5B undergo activation (Dentelli et al., 1999), endothelial cells kept in suspension or adherent to FN were evaluated for STAT5A and STAT5B activation in response to IL-3. The results presented in Fig. 6 C show that the addition of IL-3 to adherent cells induced an increase in STAT5A phosphorylation, quantified by densitometric analysis (Fig. 6 C, top). Moreover, the amount of STAT5A coimmunoprecipitated with IL-3R β common was also increased in IL-3–treated cells (unpublished data), suggesting that the activation of STAT5A in the IL-3–mediated signaling is the result of a cooperative effect between adhesion and soluble ligand.

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