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Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development.

Colognato H, Ramachandrappa S, Olsen IM, ffrench-Constant C - J. Cell Biol. (2004)

Bottom Line: Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling.Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation).However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge CB21QP, England, UK. colognato@pharm.sunysb.edu

ABSTRACT
Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.

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siRNA depletes Src family kinases in primary oligodendrocytes. (A) Src family kinase (SFK) protein expression in oligodendrocytes differentiated for 1, 2, 4, or 6 d after growth factor withdrawal. (B) SFK Western blots of G418-selected progenitors transfected with Fyn and Lyn depletion constructs. (C) Western blots of oligodendrocyte progenitors transfected with Src depletion construct. (A–C) Blots were also probed with actin antibodies as protein loading controls. (D) Immunostaining to visualize Fyn protein in a mixed population containing YFP+-transfected cells. Control (left) and Fyn(−) (middle) micrographs depict newly differentiated oligodendrocytes labeled with antibodies against Fyn (red) and GFP (green) (arrowheads). Double-labeled cells do not appear in the Fyn(−)-transfected population. (right) Control cells expressing late differentiation markers MBP (red) and YFP (green) (arrows).
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fig1: siRNA depletes Src family kinases in primary oligodendrocytes. (A) Src family kinase (SFK) protein expression in oligodendrocytes differentiated for 1, 2, 4, or 6 d after growth factor withdrawal. (B) SFK Western blots of G418-selected progenitors transfected with Fyn and Lyn depletion constructs. (C) Western blots of oligodendrocyte progenitors transfected with Src depletion construct. (A–C) Blots were also probed with actin antibodies as protein loading controls. (D) Immunostaining to visualize Fyn protein in a mixed population containing YFP+-transfected cells. Control (left) and Fyn(−) (middle) micrographs depict newly differentiated oligodendrocytes labeled with antibodies against Fyn (red) and GFP (green) (arrowheads). Double-labeled cells do not appear in the Fyn(−)-transfected population. (right) Control cells expressing late differentiation markers MBP (red) and YFP (green) (arrows).

Mentions: Oligodendrocytes have been shown previously to express three different SFKs: Fyn, Lyn, and Src (Umemori et al., 1992; Sperber et al., 2001). We confirmed that primary rat oligodendrocytes expressed these kinases, and that their expression was regulated in differentiating cells (Fig. 1 A). Progenitor cells were withdrawn at day 0 from growth factors that promote their division and prevent differentiation, and lysates were prepared from cells grown in conditions that promote oligodendrocyte differentiation, at days 1, 2, 4, or 6. Fyn and Lyn showed the highest levels of expression at days 2 and 4 of differentiation, correlating with the transition from oligodendrocyte progenitors to newly formed oligodendrocytes. Src expression was relatively difficult to detect, except at earlier stages (days 1 and 2), and was undetectable by day 6.


Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development.

Colognato H, Ramachandrappa S, Olsen IM, ffrench-Constant C - J. Cell Biol. (2004)

siRNA depletes Src family kinases in primary oligodendrocytes. (A) Src family kinase (SFK) protein expression in oligodendrocytes differentiated for 1, 2, 4, or 6 d after growth factor withdrawal. (B) SFK Western blots of G418-selected progenitors transfected with Fyn and Lyn depletion constructs. (C) Western blots of oligodendrocyte progenitors transfected with Src depletion construct. (A–C) Blots were also probed with actin antibodies as protein loading controls. (D) Immunostaining to visualize Fyn protein in a mixed population containing YFP+-transfected cells. Control (left) and Fyn(−) (middle) micrographs depict newly differentiated oligodendrocytes labeled with antibodies against Fyn (red) and GFP (green) (arrowheads). Double-labeled cells do not appear in the Fyn(−)-transfected population. (right) Control cells expressing late differentiation markers MBP (red) and YFP (green) (arrows).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172535&req=5

fig1: siRNA depletes Src family kinases in primary oligodendrocytes. (A) Src family kinase (SFK) protein expression in oligodendrocytes differentiated for 1, 2, 4, or 6 d after growth factor withdrawal. (B) SFK Western blots of G418-selected progenitors transfected with Fyn and Lyn depletion constructs. (C) Western blots of oligodendrocyte progenitors transfected with Src depletion construct. (A–C) Blots were also probed with actin antibodies as protein loading controls. (D) Immunostaining to visualize Fyn protein in a mixed population containing YFP+-transfected cells. Control (left) and Fyn(−) (middle) micrographs depict newly differentiated oligodendrocytes labeled with antibodies against Fyn (red) and GFP (green) (arrowheads). Double-labeled cells do not appear in the Fyn(−)-transfected population. (right) Control cells expressing late differentiation markers MBP (red) and YFP (green) (arrows).
Mentions: Oligodendrocytes have been shown previously to express three different SFKs: Fyn, Lyn, and Src (Umemori et al., 1992; Sperber et al., 2001). We confirmed that primary rat oligodendrocytes expressed these kinases, and that their expression was regulated in differentiating cells (Fig. 1 A). Progenitor cells were withdrawn at day 0 from growth factors that promote their division and prevent differentiation, and lysates were prepared from cells grown in conditions that promote oligodendrocyte differentiation, at days 1, 2, 4, or 6. Fyn and Lyn showed the highest levels of expression at days 2 and 4 of differentiation, correlating with the transition from oligodendrocyte progenitors to newly formed oligodendrocytes. Src expression was relatively difficult to detect, except at earlier stages (days 1 and 2), and was undetectable by day 6.

Bottom Line: Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling.Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation).However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge CB21QP, England, UK. colognato@pharm.sunysb.edu

ABSTRACT
Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.

Show MeSH
Related in: MedlinePlus