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
), a recognized STAT-binding site (1
), as detected by EMSA (Fig. 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
). In contrast, no SIF A, and little SIF C activity was detected in nuclear extracts obtained from unmanipulated B-2 cells.
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 (more ...)
The B-1 cell complexes were competed by unlabeled SIE-containing oligonucleotide but not by the consensus binding site for the nuclear factor of activated T cells (NF-AT) (Fig. B
), indicating that these complexes are specific for the SIE. The constitutive expression of SIF complexes in B-1 cells was not the result of serum induction in vitro, as these complexes were still formed from nuclear extracts prepared from B-1 cells purified and cultured in serum-free medium and were not inducible upon subsequent serum treatment (Fig. C
). Antibody to the NH2
-terminal region of STAT1α (anti-p91N), which has been shown to recognize STAT3 induced by ciliary neurotrophic factor (CNTF) (31
), disrupted SIF A induced by IL-6 in B cells (Fig. A
). The constitutively expressed B-1 SIF A–like complex was also disrupted by anti-p91N but not by an antibody to phosphotyrosine701
STAT1 (Fig. A
). Anti-p91N formed supershifted complexes with SIF A in both IL-6–stimulated B cells and untreated B-1 cells which were apparent in longer exposures and showed identical electrophoretic mobilities; antisera to STAT 4, 5, or 6 failed to react with the B-1 cell SIF A complex in EMSA supershift assays or with B-1 cell nuclear extracts in Western blotting experiments, and antibody that supershifted STAT1 activated by IFN-γ immunoinhibited/supershifted only a small amount of the SIF C complex of B-1 cells (data not shown). Because STAT3 is only known to form homodimers or to heterodimerize with STAT1, these results strongly suggest that B-1 cells differ from B-2 cells in the basal nuclear expression of STAT3 homodimers that comprise the SIF A nucleoprotein complex.
Figure 2 Immunoreactivity of B-1 cell SIF A with STAT3-specific antisera, and detection of constitutively expressed B-1 cell STAT 1 and 3 isoforms. (A) EMSA supershift/immunoinhibition analysis was performed using nuclear extracts from untreated B-1 cells ( (more ...)
To assess the contribution of phosphorylated STAT3 to the SIF A complex constitutively present in B-1 cells, we performed Shift–Western experiments, using nuclear extracted protein, the SIE-containing oligonucleotide, and antiphosphotyrosine705STAT3. The mobility shift assay is shown in Fig. B, top. Nuclear extracts from either unstimulated B-1 or B-2 cells or from B-2 cells treated with IL-6 for 15 min were incubated with the SIE oligonucleotide before EMSA. The SIF A complexes constitutively present in untreated B-1 cells and IL-6–treated B-2 cells were found to contain phosphotyrosine705STAT3, as determined by immunoblotting material obtained from the native gel (Fig. B, bottom). As a control, phosphotyrosine705STAT3 electrophoresed in the absence of the SIE-containing oligonucleotide (no DNA control) was observed to migrate more slowly in the gel, distinguishing it from SIE-bound STAT3 (data not shown). These results confirm that STAT3 is constitutively present in B-1 nuclei, as indicated by the supershifting experiments outlined above and show that it is present as a tyrosine phosphorylated protein in the SIF A complex.
We further examined the phosphorylation status of STAT3 in nuclear extracts from B-1 and B-2 cells. CNTF has been reported to induce the tyrosine phosphorylation of two STAT3 isoforms, p88 and p89 (31
). These isoforms, whose phosphorylation is also inducible by IL-6 (29
), have been termed STAT3f
(p88), for faster migrating and STAT3s
(p89), for slower migrating (32
). Immunoblotting with antiserum specific for STAT3 phosphorylated on tyrosine705
showed that B-2 cells express little nuclear phosphotyrosine705
, as expected on the basis of EMSA analysis (Fig. A
). In comparison, B-1 cell nuclear extracts were found to contain roughly equal levels of phosphotyrosine705
, respectively, which co-migrated with IL-6–induced phosphotyrosine705
STAT3 from B-2 cells (Fig. C
). In addition, immunoblotting untreated B-1 cell nuclear extracts with antibody specific for STAT1 phosphorylated on tyrosine701
) detected activated STAT1 of the p91 isoform, which was not present in nuclear extracts from untreated B-2 cells but was inducible by IFN-γ treatment (Fig. D
). Thus, the B-1–specific expression of SIF A correlates with the presence of the phosphotyrosine705
forms of STAT3s
in nuclei from unstimulated B-1 cells, and B-1 cells also constitutively express a small amount of activated p91-STAT1.
The presence of SIF A and phosphotyrosine705STAT3 in B-1 cells could not be due to macrophage contamination, because purified macrophages isolated by adherence during B cell purification from the same animals did not contain nuclear SIF A or phosphotyrosine705STAT3, and histologic examination of B-1 populations revealed less than 2% macrophage contamination (data not shown). Further, overnight incubation of B-1 cells with neutralizing antibody to IL-10 before preparation of nuclear extracts did not result in diminution of the SIF A complex observed by EMSA (data not shown), suggesting that an IL-10 autocrine loop does not account for the observed elevated levels of STAT3 in unstimulated B-1 cells and that constitutive STAT3 expression is intrinsic to this population.
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. A
). As with B-1 SIF A, anti-p91N but not antiserum to phosphotyrosine701
STAT1 disrupted SIF A induced by anti-Ig in B-2 cells (Fig. 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, phosphotyrosine705
was detected by immunoblotting using nuclear extracts from B-2 cells treated for 3 h with anti-Ig, although little phosphotyrosine705
was observed (Fig. A
). B cells stimulated with anti-Ig for less than 3 h did not contain nuclear phosphotyrosine705
(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 phosphotyrosine705
STAT3 profile of unstimulated B-1 cells, in which levels of STAT3s
are nearly equal (Fig. 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.
Figure 3 Inducible nuclear expression of STAT3 in B-2 cells treated with anti-Ig. (A) Delayed nuclear expression of SIF A in anti-Ig–stimulated B-2 cells. EMSA analysis for SIE-binding activity was carried out using nuclear extracts prepared from B-2 (more ...)
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 (more ...)
The formation and transcriptional activity of cytokineinduced STAT3 complexes have been shown to require inducible serine phosphorylation (32
). Because many sIg triggered downstream events are PKC dependent, we tested whether anti-Ig induction of STAT3 in B-2 cells is sensitive to inhibition of serine/threonine phosphorylation, using the inhibitor, H7. The induction of phosphotyrosine705
by anti-Ig was completely inhibited by H7 but not by treatment with (the control analog) HA1004 (Fig. A
). Preincubation with H7 but not with HA1004 also completely blocked the formation of the SIF A–binding complex in EMSA experiments conducted using nuclear extracts from B-2 cells stimulated with anti-Ig for 3 h (Fig. B
). These results suggest that nuclear localization of phosphotyrosine705
and the appearance of nuclear SIF A in B-2 cells stimulated by anti-Ig requires serine/ threonine phosphorylation and further implicate STAT3s
in the composition of the anti-Ig–induced SIF A complex.
The delayed tyrosine phosphorylation of STAT3 after sIg ligation in B-2 cells suggested that the synthesis of an intermediary protein is required for this response. To test this possibility, B-2 cells were stimulated with anti-Ig in the presence of the protein synthesis inhibitor cycloheximide (CHX) and nuclear extracts were prepared. CHX completely blocked the induction of phosphotyrosine705STAT3s in nuclear extracts from B-2 cells treated with anti-Ig for 3 h, whereas CHX had no effect on phosphotyrosine705STAT3 stimulated by IL-6 (Fig. C). CHX also abrogated the formation of the SIF A–binding complex in EMSA experiments performed using nuclear extracts from B-2 cells stimulated with anti-Ig for 3 h (data not shown). Thus, de novo protein synthesis is required for induction of both SIF A and of phosphotyrosine705STAT3s by anti-Ig.
Since anti-Ig is a mitogenic stimulus for B-2 cells, we reasoned that induction of STAT3 via this novel mechanism may be sensitive to immunosuppressive drugs that inhibit B cell proliferation, such as cyclosporin A, FK506, and rapamycin (37
). Immunoblot analysis of nuclear extracted protein showed substantial inhibition of anti- Ig–induced phosphotyrosine705
by rapamycin (Fig. D
). Rapamycin also significantly blocked the formation of the anti-Ig–inducible SIF A complex (Fig. E
). This effect of rapamycin is specific for sIg-triggered STAT3 because induction of phosphotyrosine705
STAT3 by IL-6 was not affected by rapamycin (data not shown). Further, B-2 cell treatment with CsA had a minimal effect on nuclear expression of phosphotyrosine705
after anti-Ig stimulation but completely inhibited nuclear phosphotyrosine705
induced by the combination of PMA and the calcium ionophore, ionomycin (data not shown) demonstrating an additional level of specificity for the effect of rapamycin on anti-Ig–induced STAT3.
Both the delayed appearance and dependence on protein synthesis of phosphotyrosine705STAT3 in B-2 cells after anti-Ig stimulation raised the possibility that sIg-mediated STAT3 induction may be due to the release of cytokines from the B cells themselves or from other contaminating cells in the B-2 cell preparation after treatment with antiIg. To address this question, nuclear extracts from the mature B cell line BAL-17 were prepared and immunoblotted for phosphotyrosine705STAT3 after stimulation with anti-Ig. Phosphotyrosine705STAT3s was induced in BAL-17 B cells by anti-Ig treatment with similar kinetics to that observed in B-2 cells (data not shown) ruling out a role for a factor secreted by a contaminating non–B cell. In addition, culture supernatants from B cells stimulated by anti-Ig for 3 h were transfered to naive cells, from which nuclear extracts were prepared after 15 min and tested for the presence of phosphotyrosine705STAT3 by immunoblotting. Supernatants from cultures treated with anti-Ig for 3 h did not induce appreciable rapid tyrosine phosphorylation of STAT3 in previously naive cells (data not shown), as would be expected of a cytokine-mediated response. These results, coupled with the sensitivity of this response to rapamycin, which does not inhibit cytokine-mediated STAT signaling, suggest that the delayed tyrosine phosphorylation of STAT3 is specific to anti-Ig treatment and is not the result of cytokine release or synthesis triggered by cell activation.
Prolonged exposure of B-2 cells to anti-Ig (e.g., for 2.5 d) has been shown to result in the acquisition of surface CD5 expression and proliferative responsiveness to PMA (39
). The possibility that prolonged sIg crosslinking produces a B-1–like basal level of nuclear activated STAT3 was tested by treating B-2 cells with anti-Ig for several days before nuclear extraction. Although B-2 cells treated with anti-Ig for 2.5 d responded to PMA by cell cycle progression to S phase, sIg-mediated nuclear SIF A (which was apparent at 3 h) had disappeared by this time (Fig. ). This result indicates that STAT3 induced by anti-Ig in B-2 cells is only transiently expressed, and thus long term T cell–independent type II (TI-2) antigenic stimulation of B-2 cells does not recapitulate the profile of activated STAT3 characteristic of B-1 cells, despite inducing other B-1–like changes. These results suggest that activated STAT3 expression is an intrinsic and unique characteristic of B-1 cells.
Figure 5 Failure of prolonged anti-Ig treatment of B-2 cells to reproduce the B-1 cell nuclear STAT3 profile. Nuclear extracts were prepared from untreated B-1 cells and from B-2 cells incubated with either medium alone (−), or treated with IL-6 for (more ...)
In conclusion, we have identified constitutive nuclear activated STAT3 in normal murine B-1 lymphocytes, representing the first nuclear transcriptional identifier for this developmentally regulated B cell population. The B-1 cell subset has been linked to spontaneously arising B cell tumors, and STAT3 has been found to be activated in v-abl
-transformed B cells, HTLV-I–transformed T cells, and v-src
– transformed fibroblasts (17
). Basal levels of nuclear phosphorylated STAT3 may reflect, or may cause, the activated state of B-1 cells, and may contribute to the self-renewing growth characteristics and the oncogenic potential of normal B-1 cells in vivo. In contrast, both egr-1
mRNA levels do not differ between B-1 and B-2 cells (41
There has been considerable debate over whether B-1 cells are derived from a separate lineage of progenitor cells or represent B-2 cells that have undergone internal biochemical and external cell surface marker changes due to prior activational or differentiative responses, such as those delivered by TI-2 antigens (42
). Our data suggest that one activity of sIg cross-linking in conventional B cells is to activate STAT3, which occurs in delayed fashion, involves phosphorylation of tyrosine705
, and is dependent upon de novo protein synthesis, serine/threonine phosphorylation, and the participation of a rapamycin-inhibitable kinase. Thus, the B cell antigen receptor is coupled to nuclear expression of activated STAT proteins (this work and references 16
). Notably, many features of this coupling stand in stark contrast to the accepted paradigm for STAT activation mediated by cytokine receptors, in which STAT phosphorylation occurs rapidly, does not require protein synthesis, and is independent of rapamycin-sensitive kinase activity. However, the basal presence of both activated STAT3s
in unstimulated B-1 cells contrasts with the transient induction of predominantly the STAT3s
isoform in anti-Ig stimulated B-2 cells and suggests that crosslinking sIg alone does not result in similar nuclear expression, in B-2 cells, of this activated transcription factor present in B-1 cells. Therefore, constitutive B-1 cell STAT3 expression suggests that the development of these B cells cannot be explained by TI-2 antigen–mediated influences alone, and that STAT proteins play a role in directing the unique behavioral and phenotypic characteristics of this population of normal cells.