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Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.

Karras JG, Wang Z, Huo L, Howard RG, Frank DA, Rothstein TL - J. Exp. Med. (1997)

Bottom Line: Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors.The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells.Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University Medical Center, Boston, Massachusetts 02118, USA.

ABSTRACT
Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

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Unique features of  the surface Ig-mediated STAT3signaling pathway. (A and B) Induction of tyrosine phosphorylated STAT3 by anti-Ig in B-2  cells requires serine/threonine  phosphorylation. Nuclear extracts were obtained from B-2  cells stimulated for 3 h with  F(ab′)2 GaMIgM (aIg) alone or  with aIg in the presence or absence of either the serine/threonine kinase inhibitor H-7, or its  less active structural analogue,  HA1004 (both at 25 μM; LC  Laboratories) and probed with  antibody specific for phosphotyrosine705STAT3 (A). Arrows indicate fast (p88) and slow  (p89) migrating forms of phosphorylated STAT3. To test for  equal loading of lanes, the blot  was reprobed with an antibody to the constitutively expressed nuclear  transcription factor, CREB. Nuclear extracts were also analyzed by  EMSA as described in Fig. 1 (B). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C. (C) Induction of STAT3s after anti-Ig treatment is blocked by cycloheximide. Primary B cells were  incubated in medium alone (M) or were treated with IL-6 (1,000 U/ml)  for 15 min, or with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for 3 h,  after which nuclear extracts were prepared. Before stimulation, some B  cell cultures were pretreated for 30 min with CHX (10 μg/ml) as indicated. Nuclear extracted protein was size separated by SDS-PAGE on  7.5% gels followed by immunoblotting with phosphotyrosine705STAT3– specific antibody, detected by ECL. Arrows indicate fast (p88) and slow  (p89) migrating forms of phosphorylated STAT3. (D and E) Rapamycin  inhibits anti-Ig–induced activation of STAT3. Primary B cells (1.5 × 107)  were incubated in medium alone (−) or were stimulated with F(ab′)2  goat anti–mouse IgM (15 μg/ml; aIg) for the indicated times, after which  nuclear extracts were prepared. Before stimulation, some B cell cultures  were pretreated for 15 min with either rapamycin at 20 ng/ml (rapa) or  ethanol (vehicle, VH). Nuclear extracted protein was size separated by  SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3–specific antibody, detected by ECL (D). The positions of molecular size markers and phospho-STAT3 (arrow) are indicated.  Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (E).  Arrows indicate positions of nucleoprotein complexes containing SIF A,  B, and C.
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Figure 4: Unique features of the surface Ig-mediated STAT3signaling pathway. (A and B) Induction of tyrosine phosphorylated STAT3 by anti-Ig in B-2 cells requires serine/threonine phosphorylation. Nuclear extracts were obtained from B-2 cells stimulated for 3 h with F(ab′)2 GaMIgM (aIg) alone or with aIg in the presence or absence of either the serine/threonine kinase inhibitor H-7, or its less active structural analogue, HA1004 (both at 25 μM; LC Laboratories) and probed with antibody specific for phosphotyrosine705STAT3 (A). Arrows indicate fast (p88) and slow (p89) migrating forms of phosphorylated STAT3. To test for equal loading of lanes, the blot was reprobed with an antibody to the constitutively expressed nuclear transcription factor, CREB. Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (B). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C. (C) Induction of STAT3s after anti-Ig treatment is blocked by cycloheximide. Primary B cells were incubated in medium alone (M) or were treated with IL-6 (1,000 U/ml) for 15 min, or with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for 3 h, after which nuclear extracts were prepared. Before stimulation, some B cell cultures were pretreated for 30 min with CHX (10 μg/ml) as indicated. Nuclear extracted protein was size separated by SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3– specific antibody, detected by ECL. Arrows indicate fast (p88) and slow (p89) migrating forms of phosphorylated STAT3. (D and E) Rapamycin inhibits anti-Ig–induced activation of STAT3. Primary B cells (1.5 × 107) were incubated in medium alone (−) or were stimulated with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for the indicated times, after which nuclear extracts were prepared. Before stimulation, some B cell cultures were pretreated for 15 min with either rapamycin at 20 ng/ml (rapa) or ethanol (vehicle, VH). Nuclear extracted protein was size separated by SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3–specific antibody, detected by ECL (D). The positions of molecular size markers and phospho-STAT3 (arrow) are indicated. Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (E). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C.

Mentions: Previous work has shown that anti-Ig treatment of B-2 cells results in STAT1 activation (35). It has been suggested that B-1 cells represent a population of conventional B cells previously activated through their antigen receptors. For these reasons, STAT3 activation was evaluated in nuclear extracts from B-2 cells treated with anti-Ig. Nuclear extracts from anti-Ig–treated B-2 cells formed a SIF A complex with the SIE similar to that observed in untreated B-1 cells, although this activity was only present in extracts from cells stimulated for 3 h or more (Fig. 3 A). As with B-1 SIF A, anti-p91N but not antiserum to phosphotyrosine701STAT1 disrupted SIF A induced by anti-Ig in B-2 cells (Fig. 3 B) and formed a supershifted complex with SIF A that was visible in longer exposures and co-migrated with a similar complex recognized by anti-p91N in IL-6–treated B cells (data not shown). Further, phosphotyrosine705STAT3s was detected by immunoblotting using nuclear extracts from B-2 cells treated for 3 h with anti-Ig, although little phosphotyrosine705STAT3f was observed (Fig. 4 A). B cells stimulated with anti-Ig for less than 3 h did not contain nuclear phosphotyrosine705STAT3s or STAT3f (data not shown). These results suggest that cross-linking sIg in B-2 cells generates activated nuclear STAT3 of predominantly the STAT3s isoform, much like IL-6 treatment of B-2 cells but dissimilar from the phosphotyrosine705STAT3 profile of unstimulated B-1 cells, in which levels of STAT3s and STAT3f are nearly equal (Fig. 2 C). Thus, constitutively expressed B-1 cell STAT3 is not the same as STAT3 induced in B-2 cells by sIg or cytokine receptor engagement.


Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.

Karras JG, Wang Z, Huo L, Howard RG, Frank DA, Rothstein TL - J. Exp. Med. (1997)

Unique features of  the surface Ig-mediated STAT3signaling pathway. (A and B) Induction of tyrosine phosphorylated STAT3 by anti-Ig in B-2  cells requires serine/threonine  phosphorylation. Nuclear extracts were obtained from B-2  cells stimulated for 3 h with  F(ab′)2 GaMIgM (aIg) alone or  with aIg in the presence or absence of either the serine/threonine kinase inhibitor H-7, or its  less active structural analogue,  HA1004 (both at 25 μM; LC  Laboratories) and probed with  antibody specific for phosphotyrosine705STAT3 (A). Arrows indicate fast (p88) and slow  (p89) migrating forms of phosphorylated STAT3. To test for  equal loading of lanes, the blot  was reprobed with an antibody to the constitutively expressed nuclear  transcription factor, CREB. Nuclear extracts were also analyzed by  EMSA as described in Fig. 1 (B). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C. (C) Induction of STAT3s after anti-Ig treatment is blocked by cycloheximide. Primary B cells were  incubated in medium alone (M) or were treated with IL-6 (1,000 U/ml)  for 15 min, or with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for 3 h,  after which nuclear extracts were prepared. Before stimulation, some B  cell cultures were pretreated for 30 min with CHX (10 μg/ml) as indicated. Nuclear extracted protein was size separated by SDS-PAGE on  7.5% gels followed by immunoblotting with phosphotyrosine705STAT3– specific antibody, detected by ECL. Arrows indicate fast (p88) and slow  (p89) migrating forms of phosphorylated STAT3. (D and E) Rapamycin  inhibits anti-Ig–induced activation of STAT3. Primary B cells (1.5 × 107)  were incubated in medium alone (−) or were stimulated with F(ab′)2  goat anti–mouse IgM (15 μg/ml; aIg) for the indicated times, after which  nuclear extracts were prepared. Before stimulation, some B cell cultures  were pretreated for 15 min with either rapamycin at 20 ng/ml (rapa) or  ethanol (vehicle, VH). Nuclear extracted protein was size separated by  SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3–specific antibody, detected by ECL (D). The positions of molecular size markers and phospho-STAT3 (arrow) are indicated.  Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (E).  Arrows indicate positions of nucleoprotein complexes containing SIF A,  B, and C.
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Figure 4: Unique features of the surface Ig-mediated STAT3signaling pathway. (A and B) Induction of tyrosine phosphorylated STAT3 by anti-Ig in B-2 cells requires serine/threonine phosphorylation. Nuclear extracts were obtained from B-2 cells stimulated for 3 h with F(ab′)2 GaMIgM (aIg) alone or with aIg in the presence or absence of either the serine/threonine kinase inhibitor H-7, or its less active structural analogue, HA1004 (both at 25 μM; LC Laboratories) and probed with antibody specific for phosphotyrosine705STAT3 (A). Arrows indicate fast (p88) and slow (p89) migrating forms of phosphorylated STAT3. To test for equal loading of lanes, the blot was reprobed with an antibody to the constitutively expressed nuclear transcription factor, CREB. Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (B). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C. (C) Induction of STAT3s after anti-Ig treatment is blocked by cycloheximide. Primary B cells were incubated in medium alone (M) or were treated with IL-6 (1,000 U/ml) for 15 min, or with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for 3 h, after which nuclear extracts were prepared. Before stimulation, some B cell cultures were pretreated for 30 min with CHX (10 μg/ml) as indicated. Nuclear extracted protein was size separated by SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3– specific antibody, detected by ECL. Arrows indicate fast (p88) and slow (p89) migrating forms of phosphorylated STAT3. (D and E) Rapamycin inhibits anti-Ig–induced activation of STAT3. Primary B cells (1.5 × 107) were incubated in medium alone (−) or were stimulated with F(ab′)2 goat anti–mouse IgM (15 μg/ml; aIg) for the indicated times, after which nuclear extracts were prepared. Before stimulation, some B cell cultures were pretreated for 15 min with either rapamycin at 20 ng/ml (rapa) or ethanol (vehicle, VH). Nuclear extracted protein was size separated by SDS-PAGE on 7.5% gels followed by immunoblotting with phosphotyrosine705STAT3–specific antibody, detected by ECL (D). The positions of molecular size markers and phospho-STAT3 (arrow) are indicated. Nuclear extracts were also analyzed by EMSA as described in Fig. 1 (E). Arrows indicate positions of nucleoprotein complexes containing SIF A, B, and C.
Mentions: Previous work has shown that anti-Ig treatment of B-2 cells results in STAT1 activation (35). It has been suggested that B-1 cells represent a population of conventional B cells previously activated through their antigen receptors. For these reasons, STAT3 activation was evaluated in nuclear extracts from B-2 cells treated with anti-Ig. Nuclear extracts from anti-Ig–treated B-2 cells formed a SIF A complex with the SIE similar to that observed in untreated B-1 cells, although this activity was only present in extracts from cells stimulated for 3 h or more (Fig. 3 A). As with B-1 SIF A, anti-p91N but not antiserum to phosphotyrosine701STAT1 disrupted SIF A induced by anti-Ig in B-2 cells (Fig. 3 B) and formed a supershifted complex with SIF A that was visible in longer exposures and co-migrated with a similar complex recognized by anti-p91N in IL-6–treated B cells (data not shown). Further, phosphotyrosine705STAT3s was detected by immunoblotting using nuclear extracts from B-2 cells treated for 3 h with anti-Ig, although little phosphotyrosine705STAT3f was observed (Fig. 4 A). B cells stimulated with anti-Ig for less than 3 h did not contain nuclear phosphotyrosine705STAT3s or STAT3f (data not shown). These results suggest that cross-linking sIg in B-2 cells generates activated nuclear STAT3 of predominantly the STAT3s isoform, much like IL-6 treatment of B-2 cells but dissimilar from the phosphotyrosine705STAT3 profile of unstimulated B-1 cells, in which levels of STAT3s and STAT3f are nearly equal (Fig. 2 C). Thus, constitutively expressed B-1 cell STAT3 is not the same as STAT3 induced in B-2 cells by sIg or cytokine receptor engagement.

Bottom Line: Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors.The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells.Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University Medical Center, Boston, Massachusetts 02118, USA.

ABSTRACT
Cytokine and growth factor receptor engagement leads to the rapid phosphorylation and activation of latent, cytosolic signal transducers and activators of transcription (STAT) proteins, which then translocate to the nucleus where they regulate transcriptional events from specific promoter sequences. STAT3 expression in particular has been associated with Abl, Src, and HTLV-1 transformation of normal cells. B-1 lymphocytes are self-renewing, CD5+ B cells that display a propensity for malignant transformation and are the normal counterpart to human chronic lymphocytic leukemias. Further, B-1 cells are characterized by aberrant intracellular signaling, including hyperresponsiveness to phorbol ester PKC agonists. Here we demonstrate that B-1 lymphocytes constitutively express nuclear activated STAT3, which is not expressed by unmanipulated conventional (B-2) lymphocytes. In contrast, STAT3 activation is induced in B-2 cells after antigen receptor engagement in a delayed fashion (after 3 h). Induction of STAT3 is inhibited by both the serine/threonine protein kinase inhibitor H-7 and the immunosuppressive drug rapamycin and requires de novo protein synthesis, demonstrating novel coupling between sIg and STAT proteins that differs from the classical paradigm for STAT induction by cytokine receptors. The inability of prolonged stimulation of conventional B-2 cells with anti-Ig, a treatment sufficient to induce CD5 expression, to result in sustained STAT3 activation suggests that STAT3 is a specific nuclear marker for B-1 cells. Thus, STAT3 may play a role in B cell antigen-specific signaling responses, and its constitutive activation is associated with a normal cell population exhibiting intrinsic proliferative behavior.

Show MeSH
Related in: MedlinePlus