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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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Constitutive expression of specific nuclear SIE-binding activity that co- migrates with IL-6–induced SIF A in B-1  cells. (A) EMSA analysis of nuclear extracts prepared from B-2 cells (15 × 106) incubated in  medium alone or treated with IL-6 (1,000 U/ ml for 15 min; Genzyme, Cambridge, MA) or  IFN-γ (γ; 5 ng/ml for 15 min; Genzyme), or  B-1 cells (7.5 × 106) incubated in medium  alone. Nuclear extracts were incubated with  radiolabeled oligonucleotide containing the  high-affinity c-fos SIE sequence (28). Arrows  indicate the positions of STAT3 homodimers  (SIF A), STAT3–STAT1 heterodimers (SIF  B), and STAT1 homodimers (SIF C). (B)  Specific binding activity of the constitutive B-1  SIE-binding complex. Competition analysis  was performed on nuclear extracts from untreated B-1 cells in an EMSA using 20-fold  excess unlabeled SIE or NF-AT (control) oligonucleotides. Arrows indicate the positions  of SIE-binding complexes containing SIF A,  B, and C. (C) Serum does not induce SIF A  in B-1 cells. SIE–EMSA analysis of nuclear  extracts prepared from B-1 cells purified and  cultured in the presence of serum-free medium alone (lane 1), or in serum-free medium  plus IL-6 (1,000 U/ml for 15 min; lane 3), or  subsequent resuspension in serum-containing  RPMI medium for 15 min (lane 4). B-2 cells  (lane 2) were cultured in serum-containing  RPMI medium alone.
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Figure 1: Constitutive expression of specific nuclear SIE-binding activity that co- migrates with IL-6–induced SIF A in B-1 cells. (A) EMSA analysis of nuclear extracts prepared from B-2 cells (15 × 106) incubated in medium alone or treated with IL-6 (1,000 U/ ml for 15 min; Genzyme, Cambridge, MA) or IFN-γ (γ; 5 ng/ml for 15 min; Genzyme), or B-1 cells (7.5 × 106) incubated in medium alone. Nuclear extracts were incubated with radiolabeled oligonucleotide containing the high-affinity c-fos SIE sequence (28). Arrows indicate the positions of STAT3 homodimers (SIF A), STAT3–STAT1 heterodimers (SIF B), and STAT1 homodimers (SIF C). (B) Specific binding activity of the constitutive B-1 SIE-binding complex. Competition analysis was performed on nuclear extracts from untreated B-1 cells in an EMSA using 20-fold excess unlabeled SIE or NF-AT (control) oligonucleotides. Arrows indicate the positions of SIE-binding complexes containing SIF A, B, and C. (C) Serum does not induce SIF A in B-1 cells. SIE–EMSA analysis of nuclear extracts prepared from B-1 cells purified and cultured in the presence of serum-free medium alone (lane 1), or in serum-free medium plus IL-6 (1,000 U/ml for 15 min; lane 3), or subsequent resuspension in serum-containing RPMI medium for 15 min (lane 4). B-2 cells (lane 2) were cultured in serum-containing RPMI medium alone.

Mentions: To determine whether the unusual growth characteristics of B-1 cells are accompanied by differences in the regulation of STAT proteins, we compared STAT DNA-binding activities between resting B-1 and B-2 cells. Nuclear extracts from untreated B-1 cells formed protein–DNA complexes with the high-affinity sis-inducible element (SIE) of the c-fos gene (27), a recognized STAT-binding site (1), as detected by EMSA (Fig. 1 A). The major B-1 cell–specific SIE-binding activity was observed to co-migrate with the IL-6–stimulated sis-inducible factor (SIF) A binding complex, with a smaller amount co-migrating similarly to the IFN-γ–induced SIF C complex (24, 28–30). In contrast, no SIF A, and little SIF C activity was detected in nuclear extracts obtained from unmanipulated B-2 cells.


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)

Constitutive expression of specific nuclear SIE-binding activity that co- migrates with IL-6–induced SIF A in B-1  cells. (A) EMSA analysis of nuclear extracts prepared from B-2 cells (15 × 106) incubated in  medium alone or treated with IL-6 (1,000 U/ ml for 15 min; Genzyme, Cambridge, MA) or  IFN-γ (γ; 5 ng/ml for 15 min; Genzyme), or  B-1 cells (7.5 × 106) incubated in medium  alone. Nuclear extracts were incubated with  radiolabeled oligonucleotide containing the  high-affinity c-fos SIE sequence (28). Arrows  indicate the positions of STAT3 homodimers  (SIF A), STAT3–STAT1 heterodimers (SIF  B), and STAT1 homodimers (SIF C). (B)  Specific binding activity of the constitutive B-1  SIE-binding complex. Competition analysis  was performed on nuclear extracts from untreated B-1 cells in an EMSA using 20-fold  excess unlabeled SIE or NF-AT (control) oligonucleotides. Arrows indicate the positions  of SIE-binding complexes containing SIF A,  B, and C. (C) Serum does not induce SIF A  in B-1 cells. SIE–EMSA analysis of nuclear  extracts prepared from B-1 cells purified and  cultured in the presence of serum-free medium alone (lane 1), or in serum-free medium  plus IL-6 (1,000 U/ml for 15 min; lane 3), or  subsequent resuspension in serum-containing  RPMI medium for 15 min (lane 4). B-2 cells  (lane 2) were cultured in serum-containing  RPMI medium alone.
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Figure 1: Constitutive expression of specific nuclear SIE-binding activity that co- migrates with IL-6–induced SIF A in B-1 cells. (A) EMSA analysis of nuclear extracts prepared from B-2 cells (15 × 106) incubated in medium alone or treated with IL-6 (1,000 U/ ml for 15 min; Genzyme, Cambridge, MA) or IFN-γ (γ; 5 ng/ml for 15 min; Genzyme), or B-1 cells (7.5 × 106) incubated in medium alone. Nuclear extracts were incubated with radiolabeled oligonucleotide containing the high-affinity c-fos SIE sequence (28). Arrows indicate the positions of STAT3 homodimers (SIF A), STAT3–STAT1 heterodimers (SIF B), and STAT1 homodimers (SIF C). (B) Specific binding activity of the constitutive B-1 SIE-binding complex. Competition analysis was performed on nuclear extracts from untreated B-1 cells in an EMSA using 20-fold excess unlabeled SIE or NF-AT (control) oligonucleotides. Arrows indicate the positions of SIE-binding complexes containing SIF A, B, and C. (C) Serum does not induce SIF A in B-1 cells. SIE–EMSA analysis of nuclear extracts prepared from B-1 cells purified and cultured in the presence of serum-free medium alone (lane 1), or in serum-free medium plus IL-6 (1,000 U/ml for 15 min; lane 3), or subsequent resuspension in serum-containing RPMI medium for 15 min (lane 4). B-2 cells (lane 2) were cultured in serum-containing RPMI medium alone.
Mentions: To determine whether the unusual growth characteristics of B-1 cells are accompanied by differences in the regulation of STAT proteins, we compared STAT DNA-binding activities between resting B-1 and B-2 cells. Nuclear extracts from untreated B-1 cells formed protein–DNA complexes with the high-affinity sis-inducible element (SIE) of the c-fos gene (27), a recognized STAT-binding site (1), as detected by EMSA (Fig. 1 A). The major B-1 cell–specific SIE-binding activity was observed to co-migrate with the IL-6–stimulated sis-inducible factor (SIF) A binding complex, with a smaller amount co-migrating similarly to the IFN-γ–induced SIF C complex (24, 28–30). In contrast, no SIF A, and little SIF C activity was detected in nuclear extracts obtained from unmanipulated B-2 cells.

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