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Rex1 (Zfp42), first identified as a gene that is transcriptionally repressed by retinoic acid (RA), encodes a zinc finger transcription factor expressed at high levels in F9 teratocarcinoma stem cells, embryonic stem (ES) cells, and other stem cells. Loss of both alleles of Rex1 by homologous recombination alters the RA-induced differentiation of F9 cells, a model of pluripotent ES cells. We identified Suppressor of Cytokine Signaling-3 (SOCS3) as a gene which exhibits greatly increased transcriptional activation in RA, cyclicAMP and theophylline (RACT) treated F9 Rex1−/− cells (~25-fold) as compared to wild type (Wt) cells (~2.5-fold). By promoter deletion, mutation and transient transfection analyses, we have shown that this transcriptional increase is mediated by the STAT3 DNA binding elements located between −99 to −60 in the SOCS-3 promoter. Overexpression of STAT3 dominant negative mutants greatly diminishes this SOCS-3 transcriptional increase in F9 Rex1−/− cells. This increase in SOCS-3 transcription is associated with a 4–5 fold higher level of tyrosine phosphorylated STAT3 in the RACT treated F9 Rex1−/− cells as compared to Wt. Dominant negative Src tyrosine kinase, Jak2, and protein kinase A (PKA) partially reduce the transcriptional activation of the SOCS-3 gene in RACT-treated F9 Rex1 null cells. In contrast, parathyroid hormone peptide enhances the effect of RA in F9 Rex1−/− cells, but not in F9 Wt. Thus, Rex-1, which is highly expressed in stem cells, inhibits signaling via the JAK/STAT pathway, thereby modulating the differentiation of F9 cells.
Retinoids such as all-trans retinoic acid (RA) have been shown to play important roles during mammalian development and in the processes of cell differentiation in various cell types1; 2; 3; 4; 5. The diverse biological effects of RA are mediated by its ability to act as both an activator and repressor of gene transcription. Rex1 (Zfp42) (for reduced expression) was first discovered as a gene whose expression is greatly reduced upon RA treatment in F9 teratocarcinoma stem cells6, cells which resemble the pluripotent, embryonic stem (ES) cells of the inner cell mass (ICM) of the mouse blastocyst7. F9 stem cells differentiate into primitive endoderm when treated with RA, and into parietal endoderm when treated with RACT (retinoic acid plus cyclic AMP and theophylline). Rex1 encodes a protein which contains four “cys-his” zinc finger motifs, indicating that Rex1 functions as a transcription factor8; 9. When wild type F9 cells are cultured in the presence of exogenous RA and differentiate along the primitive endoderm (PrE), parietal endoderm (PE), or visceral endoderm (VE) pathways, Rex1 mRNA levels begin to decline at 12 h, and by 96 h the Rex1 mRNA level is reduced several-fold relative to the level in untreated F9 stem cells6; 10. In addition to its high level of expression in F9 stem cells, the Rex1 gene is expressed at high levels in murine ES cells10 and has been reported to be transcriptionally regulated by nanog11. Rex1 expression has frequently been used as a molecular marker of both ES and adult murine and human stem cells12; 13; 14; 15; 16; 17; 18; 19; 20. In adult male mice, the Rex1 gene is expressed in the testis at a specific stage during the process of spermatogenesis10. We've also shown that Rex1 expression is reduced in human kidney tumor specimens as compared to normal tissue21.
Current models suggest that Rex1 modifies differentiation by its effects on the transcription of target genes, though the relevant target genes haven't been described. Through the use of DNA microarray analyses (Xu and Gudas, unpublished) we have identified genes that are differentially expressed between RA-treated F9 Wt cells and F9 Rex1−/− cells, which were generated in our laboratory via homologous recombination22. One of these genes is the suppressor of cytokine signaling-3 (SOCS-3). One function of SOCS-3 is in the regulation of the JAK/STAT pathway, which is activated by several cytokines. Cytokines such as LIF (leukemia inhibitory factor) regulate cellular behavior by interacting with receptors on the plasma membrane of target cells and activating intracellular signal transduction cascades, including the JAK-STAT pathway23; 24; 25; 26. STAT proteins such as STAT3 control cell cycle progression and apoptosis, and thus overexpression or misregulation of STAT gene expression can contribute to oncogenesis27; 28; 29; 30; 31; 32; 33. Conversely, STAT3 blockade decreased tumor growth in a xenograft model of squamous carcinoma of the head and neck34 and in various human cultured cancer cell lines35. The ablation of STAT3 results in an embryonic lethal phenotype. Homozygous STAT3-null embryos degenerate rapidly immediately after blastocyst implantation36.
Among at least three classes of negative regulators of the JAK-STAT pathway is the family of SOCS proteins37; 38. SOCS-3 is a potent inhibitor of the JAK-STAT signaling cascade, negatively regulating signal transduction of a variety of cytokines39. SOCS-3 negatively regulates LIF signaling by directly binding to the JAK/LIFR/gp130 complex40, and inhibiting JAK kinase activity41. A STAT3 binding element is functionally critical for the positive transcriptional regulation of murine SOCS-3 promoter activity42, and this autoregulatory circuit between SOCS-3 and STAT3 maintains proper balance between cell renewal and cell differentiation43; 44. Although SOCS3 has primarily been considered to be a negative regulator of the JAK-STAT signaling cascade, in some cell types SOCS-3 transcription greatly increases during the differentiation process and SOCS-3 has been shown to be required for proper myoblast differentiation45. SOCS-3 gene expression is high in some tumors, including breast46 and cutaneous T-cell lymphoma47. The SOCS-3 gene is often methylated in head and neck squamous cell carcinoma48 and in hepatocellular carcinoma49. SOCS-3 overexpression in liver causes insulin resistance50 and SOCS-3 expression is induced by resistin, a hormone which antagonizes the effects of insulin in adipocytes51. SOCS-3-deficient mice die as a result of placental defects during embryonic development52; 53.
Understanding the mechanisms by which the lack of Rex1 protein results in aberrantly high SOCS-3 expression will better define the roles of Rex1 in stem cell renewal, differentiation, and early embryogenesis. In this series of experiments, we examine the mechanisms by which the murine SOCS-3 gene is transcriptionally activated in response to RA and dibutyryl cyclic AMP (db cAMP) in F9 Wt cells versus F9 Rex1−/− cells. We demonstrate that the SOCS-3 gene is induced by RA and dibutyryl cAMP via a transcriptional mechanism involving PKA, Src, and Jak2. This transcriptional activation is greatly increased in the absence of Rex1.
We used DNA microarray assays to identify Rex1 target genes by comparison of F9 Wt and F9 Rex1−/− knockout cells cultured with or without RA. From the microarray experiments (Xu and Gudas, in preparation), the murine SOCS-3 gene scored the highest level of difference, showing a 17.7-fold higher expression in RA-treated R21 Rex1−/− cells as compared to RA-treated F9 Wt cells. This high level of difference in expression and the fact that SOCS-3 is a key regulator of the STAT3 signaling pathway resulted in the selection of the SOCS-3 gene for further analysis.
F9 WT cells and two independently isolated F9 Rex1−/− knockout cell lines (R21 and R5) were cultured for 24 h, 48 h, or 72 h in the presence of 1 μM RA, 250 μM dibutyryl cAMP, and 250 μM theophylline (RACT) as previously described54; 55; 56; 57; 58. SOCS-3 mRNA was present in untreated F9 Wt cells, and RA or CT alone had only a small effect on the levels of SOCS-3 transcripts at 72 hr. (Figure 1, lower panel). The combination of RA and CT (RACT) induced the expression of SOCS-3 transcripts in F9 Wt (for 48 h: 2.43 ± 0.58 fold, n=3, p= 0.0038; for 72 h: 5.74 ± 0.53 fold, n=3, p= 0.0004) (Fig. 1).
The expression of SOCS-3 transcripts was just detectable in untreated F9 Rex1−/− (R21) cells (Fig. 1). After 48 h of RA, SOCS-3 mRNA expression was induced (for 48 h: 5.78 ± 0.83 fold, n=3, p= 0.035; for 72 h: 4.3 ± 0.53 fold, n=3, p= 0.005) (Figure 1). CT treatment alone also induced SOCS-3 expression (for 24 h: 2.32 ± 0.06 fold, n=3, p= 0.023; for 72 h: 3.32 ± 0.22 fold, n=3, p= 0.003), although the effect of CT was smaller than the effect of RA on SOCS-3. RACT treatment was able to increase SOCS-3 mRNA levels in Rex1−/− cells to a much greater degree than in F9 Wt cells (for 24 h: 6.54 ± 1.83 fold, n=3, p= 0.021; for 48 h: 25.95 ± 3.53 fold, n=3, p= 0.001; for 72 h: 16.56 ± 0.78 fold, n=3, p= 0.0005). The same results were observed in two independently isolated F9 Rex1−/− null cell lines R21 and R5) (Figure 1). Thus, RACT treatment of F9 Rex1−/− cells resulted in a ~25-fold increase in SOCS-3 transcripts at 48 hr. vs. a ~2.5-fold increase in F9 Wt.
To confirm that the increase in SOCS-3 transcripts resulted from the absence of Rex1−/−, the full-length Rex1 cDNA was stably transfected back into the F9 Rex1−/− (R21) cells (Fig. 2). In part because the exogenous Rex1 gene was driven by a SV40 promoter and SV40-driven transcription is not efficient in F9 cells59, the expression of Rex1 in the Rex1 “put-back” lines was only ~10% of its expression in F9 Wt cells. However, this low level of Rex1 mRNA was sufficient to partially reverse the expression pattern of SOCS-3 mRNA to its expression pattern in F9 Wt cells (Figure 2), suggesting that only a low level of Rex1 expression is required to reduce the level of SOCS-3 gene induction.
We also examined SOCS-3 protein levels in F9 WT, F9 Rex1−/− (R21 and R5), and the stably transfected R21JX12 cells. Cells from each cell line were cultured in the presence of 1 μM RA, 250 μM dibutyryl cAMP, and 250 μM theophylline (RACT). After 72 h, total proteins were harvested for Western analysis of SOCS-3. In F9 Wt cells the SOCS-3 protein was not expressed at a detectable level in control, RA-treated, and CT-treated cells (Figure 3), while RACT treatment resulted in induction of SOCS-3 protein expression to a limited extent (<2 fold). In the two independently-isolated F9 Rex1−/− null cell lines, SOCS-3 protein was not expressed in control cells (Figure 3). RA or CT alone did not have any effect on SOCS-3 protein expression, while RACT greatly increased the SOCS-3 protein level (Fig. 3). In the cells in which Rex1 was stably transfected back into the F9 Rex1−/− (R21) cells, the expression of the SOCS-3 protein was partially reversed to its expression pattern in F9 Wt cells (Fig. 3). Thus, the expression of SOCS-3 protein correlates with the expression of SOCS-3 transcripts.
Constructs 2, 6 and 6D2C (referred as 6D2 in the original paper)42 which use 5′ flanking DNA of the SOCS-3 gene to drive luciferase reporter expression, were provided to us by Dr. S. Melmed at Cedars-Sinai Medical Center. For measurement of SOCS-3 promoter activity, the F9 Wt cells, the two F9 Rex1−/− null lines (R21, R5), and the Rex1 stably transfected line (R21JX12) were transiently transfected with pGL3Basic alone, construct 2 (nucleotides −2757 to −714), construct 6 (nucleotides −2757 to +929), or construct 6D2C (nucleotides −2757 to +929, but with the complete tandem STAT binding region from nucleotides −99 to −60 deleted) (Figure 4A). The vehicle, pGL3Basic, was used as a control (V). Construct 2 did not contain an essential region of the minimal promoter of SOCS-3 (−714 to +929), and hence it served as another control. The transfected cells were either untreated, or treated with 1 μM RA, 250 μM dibutyryl cAMP, and 250 μM theophylline (RACT) for 48 h. Cells were then harvested for luciferase assays. The durations of the treatments were determined based on the Northern results (Figure 1) in which the expression of SOCS-3 transcript was not induced until after 48 h of RA treatment of F9 Rex1−/− cells.
In F9 Wt cells, the effects of RA, CT, and RACT on construct 6 were not significant (for all values: fold change <2, n=4, p>0.05). The deletion of the STAT3 binding repeats (construct 6D2C) did not alter the response of SOCS-3 promoter to RA, CT, and RACT treatments significantly (for all values: fold change <2, n=4, p>0.05) (Fig. 4B, inset).
In F9 Rex1−/− (R21) cells, RA addition activated the SOCS-3 promoter (construct 6) by at most two-fold (2 ± 0.19 fold, n=4, p=0.05). The addition of CT increased the SOCS-3 promoter activity greatly (18.2 ± 2.5 fold, n=4, p=0.000015). The induction of SOCS-3 promoter activity by the RACT drug combination was even greater (34.4 ± 7.8 fold, n=4, p=1.5 × 10−6) (Fig. 4B). When the STAT3 binding repeats were deleted, the effect of RA on the promoter 6D2C/reporter activity was no longer significant (1.2 ± 0.8 fold, n=4, p>0.05); the effect of CT on this SOCS-3 promoter activity was no longer significant (3.2 ± 1.1 fold, n=4, p>0.05); and the effect of RACT on construct 6D2C SOCS-3 promoter activity was reduced to an activation of only 8 ± 2.7 fold (n=4, p=0.02), which was much lower than the induction of construct 6 seen with RACT treatment (34.4 ± 7.8 fold) (Fig. 4B). Similar results were observed in the F9 Rex1−/− (R5) cells (Fig. 4B). The activities of the SOCS-3 promoter constructs in the exogenous Rex1 stably transfected (“Put-back”) cells were like those in F9 Wt cells (Fig. 4B). Similarly, SOCS-3 promoter activity decreased when increasing levels of a Rex1 expression vector were cotransfected into F9 Rex1−/− cells (Fig. 4C). Thus, the SOCS-3 promoter is substantially more active in the absence of Rex1, and re-addition of Rex1 to the Rex1−/− cells reduced SOCS-3 promoter activity.
When the tandem STAT binding sites were deleted from the 5' flanking region of murine SOCS-3 gene, the RA-, CT-, and RACT-induced activation of the SOCS-3 promoter activity decreased by more than six-fold (Fig. 4B). We hypothesized that this decrease resulted from the inability of positively acting STAT3 proteins to bind to this regulatory region of the SOCS-3 promoter. To test this hypothesis, F9 Wt cells and R21 Rex1−/− cells were co-transfected with construct 6 and a number of STAT3 expression plasmids (Fig. 5). These plasmids include the expression vector construct 6 alone (V), the WT STAT3 (STAT3), two dominant negative (DN) forms of STAT3 (Y705F, D715)60, and the constitutively active form of STAT3 (STAT3C)61. For Y705, the tyrosine residue that is phosphorylated by JAK kinases in the WT STAT3 is mutated to a phenylalanine. Therefore, Y705 can no longer be phosphorylated. D715 lacks the C-terminal transcriptional activation domain and hence, D715 can no longer transcriptionally activate target genes. Y705 is more potent than D715 as a DN mutant60. For STAT3C, an alanine residue and an asparagine residue within the C-loop of the SH2 domain were both mutated to cysteine residues. As a result of the mutations, two STAT3C molecules dimerize, bind to DNA with a greater affinity, and activate transcription persistently61; 62. The transfected cells were either untreated or treated with 1 μM RA, 250 μM db cAMP, and 250 μM theophylline (RACT) for 48 h. Then cells were harvested for luciferase assays.
In F9 Wt cells, the co-transfection of WT STAT3 and the two DN mutants of STAT3 (Y705F and D715) did not alter SOCS-3 promoter (construct 6) activity significantly (for all values: fold change < 2, n=3, p>0.05) (Figure 5). The co-transfection of the constitutively active mutant, STAT3C, induced SOCS-3 promoter activity in the control, RA-, CT-, and RACT-treated F9 Wt cells by around two-fold each (Fig. 5, inset).
In F9 Rex1−/− (R21) cells, the co-transfection of WT STAT3 (STAT3 in Fig. 5B) reduced SOCS-3 promoter activity in CT- (−2.7 ± 0.8 fold, n=3, p=0.035) and RACT-treated (−2.54 ± 0.56 fold, n=3, p=0.017) cells relative to transfection of construct 6 alone (V). There was no significant effect in control (−1.67 ± 0.2 fold, n=3, p>0.05) or RA-treated (−1.88 ± 0.76 fold, n=3, p>0.05) cells (Fig. 5). A possible explanation for this result is that the transfected WT STAT3 behaved as a mild dominant negative in the cells. It has been reported that a naturally truncated STAT3 could bind to phosphorylated STAT3 monomers and act as a dominant negative63; 64.
The co-transfection of the Y705F expression construct reduced the SOCS-3 promoter activity in CT- (−13.3 ± 3.76 fold, n=3, p=0.001) and RACT-treated cells (−24.8 ± 3.76 fold, n=3, p=0.002) relative to the transfection of construct 6 alone (V) or construct 6 plus Wt STAT3 (STAT3) (Fig. 5B). There was no significant effect in control (−2.16 ± 0.26 fold, n=3, p>0.05) or RA-treated (−2.4 ± 0.75 fold, n=3, p>0.05) cells (Fig. 5B). Similarly, the co-transfection of a construct harboring another DN mutant of STAT3, Y715, also reduced SOCS-3 promoter activities in CT- and RACT- treated cells relative to the transfection of construct 6 alone (V); this reduction was not as great as that seen with the Y705F mutant STAT3 (Fig. 5B). Thus, the large increase in SOCS-3 promoter activity in F9 Rex1−/− cells is dependent on activated STAT3.
In RACT treated F9 Wt cells co-transfection of the plasmid encoding the constitutively active mutant STAT3C resulted in a statistically significant increase in activity over that of construct 6 transfected alone (V) (Fig. 5B). In contrast, compared with the same treatments in the Rex1−/− cells transfected with only the SOCS-3 promoter/luciferase construct 6 (V in Fig. 5B), the co-transfection of the constitutively active mutant, STAT3C, greatly induced SOCS-3 promoter activity in control (5.3 ± 0.47 fold, n=3, p=0.02), RA- (13.03 ± 3.2 fold, n=3, p=0.001), and CT- treated (2.4 ± 0.13 fold, n=3, p=0.023) F9 Rex1−/− cells. Strikingly, transfection of this constitutively active STAT3C construct induced SOCS-3 promoter/reporter activity in the presence of RA alone only in the F9 Rex1−/− cells. In RACT-treated STAT3C transfected Rex1−/− cells the induction over the activity of the construct 6 transfected alone (V) was not significant (1.08 ± 0.08 fold, n=3, p>0.05) (Fig. 5B). Since RACT does not cause a further increase in the presence of constitutively activated STAT, we hypothesized that the effects of RACT are mediated by STAT3 in F9 Rex1−/− cells. To test this hypothesis, we examined how the loss of Rex1 influenced STAT3 activity.
F9 Wt and two independently-isolated F9 Rex1−/− null lines (R21 and R31) were cultured for 24 h or 72 h in the presence of 1 μM RA, 250 μM dibutyryl cAMP, and 250 μM theophylline (RACT). Total RNA was extracted for the Northern analysis of STAT3 (Figure 6A). RACT treatment of F9 Wt and the two F9 Rex1−/− cell lines resulted in the induction of STAT3 transcripts to a much greater level at 72 h (~7.5-fold). However, no differences in STAT3 mRNA expression were detected between F9 Wt cells and the two F9 Rex1−/− cell lines (Fig. 6A, lower panel for quantitation).
F9 Wt, the F9 Rex1−/− null lines R21 and R5, and the R21JX12 cell line were untreated, or treated with RA, CT, and RACT, for 48 hours. Total proteins were harvested for Western analyses of total STAT3 and phosphorylated STAT3 protein (Figure 6B). There was no difference in basal STAT3 protein expression among the four cell lines (Fig. 6B). The treatments with RA, CT, or RACT did not result in a statistically significant increase in the total STAT3 protein level in any of the four cell lines (Fig. 6B, second gel panel). Thus, total STAT3 protein levels are regulated differently, and are not correlated with STAT3 transcript levels in RACT treated F9 Wt and Rex1−/− cell lines.
Tyrosine phosphorylated STAT3 was then measured in the same experiment, and a slight increase in expression of phosphorylated STAT3 was seen in RACT-treated, but not in control, RA-, or CT-treated F9 Wt cells (Fig. 6B, top gel panel, and bar graph below for quantitation). In F9 Rex1−/− cells, RA induced the phosphorylation of STAT3, and RACT greatly increased the level of phosphorylated STAT3 (Fig. 6B, top gel panel, bar graph below for quantitation). The RACT-induced STAT3 phosphorylation in F9 Rex1−/− cells was ~4–5 fold higher than in F9 Wt cells (Fig. 6B, bar graph). In the Rex1 “put-back” stably transfected line, the expression pattern of phosphorylated STAT3 was reversed to a pattern similar to that in F9 Wt cells (Fig. 6B, top gel panel, and bar graph below for quantitation). SOCS-3 protein levels in the same cell extracts are also shown (Fig. 6B, third gel panel). Thus, the level of tyrosine phosphorylated STAT3, and, as expected, the level of SOCS-3 protein were much higher in RACT treated F9 Rex1−/− cells that in RACT treated F9 Wt cells.
Many actions of cyclic AMP are mediated by protein kinase A (PKA) (for rev.65). This serine/threonine kinase exists as an inactive tetramer composed of two regulatory and two catalytic subunits. Binding of cyclic AMP to the regulatory subunits results in the release of the active catalytic subunits and subsequent phosphorylation of target substrates66. Regulatory subunits with mutations in the cAMP-binding domains confer a dominant inhibition of the PKA system in cells67. Recent evidence suggests that cyclic AMP signaling also occurs through pathways other than the activation of PKA68.
To determine whether the action of cyclic AMP in this system was mediated by PKA, a construct encoding a PKA dominant negative (DN) mutant MT Rev(AB) (Figure 7A) was co-transfected with the SOCS-3 promoter/reporter (construct 6 from Fig. 4). After transient transfection, cells were either untreated, or treated with RA, CT, or RACT, for 48 hours. The PKA DN mutant did not significantly alter SOCS-3 promoter/reporter activity in either control or RA treated F9 Wt or Rex1−/− cells (Figure 7B). However, in CT-treated F9 Wt cells, the PKA DN mutant reduced SOCS-3 promoter/reporter activity by 3.52 ± 0.29 fold (n=3, p=0.003). In CT-treated R21 Rex1−/− cells, the PKA DN mutant reduced SOCS-3 promoter/reporter activity by 2.92 ± 0.086 fold (n=3, p=0.02). In RACT-treated F9 Wt cells, the PKA DN mutant reduced SOCS-3 promoter/reporter activity by 3.5 ± 0.46 fold (n=3, p=0.003). In RACT-treated R21 Rex1−/− cells, the PKA DN mutant reduced SOCS-3 promoter/reporter activity by 2.68 ± 0.41 fold (n=3, p=0.05). The results from these experiments indicate that treatment with CT or RACT activates SOCS-3 transcription through a PKA-dependent pathway.
STAT3 was originally identified as the acute phase response factor69 and STAT3 is tyrosine phosphorylated by JAKs following the binding of members of the IL-6 family of cytokines to their receptors. Four members of the JAK family have been identified so far: Jak1, Jak2, Jak3, and Tyk-270. Jak1, Jak2 and Tyk-2 are ubiquitous, and Jak3 is expressed primarily in T lymphocytes70. Recent studies have shown that STAT proteins can also be activated by a variety of receptor and non-receptor protein tyrosine kinases71; 72; 73; 74. These protein tyrosine kinases include epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR)75; 76; 77, colony stimulating factor-1 receptor70, and the Src tyrosine kinase78.
To determine the pathway by which STAT3 is activated and consequently, SOCS-3 transcription is activated, Jak1 DN, Jak2 DN, and Src DN mutants (Figure 8A) were co-transfected with the SOCS-3 promoter/reporter (construct 6). After transfection, cells were either untreated or treated with RACT for 48 hours. In F9 Wt cells, the Jak1 DN, Jak2 DN, and Src DN constructs did not alter the SOCS-3 promoter/reporter activity significantly in either control cells or RACT-treated cells (Figure 8B).
Jak1 DN, Jak2 DN, and Src DN mutants did not affect SOCS-3 transcription significantly in Rex1−/− cells under control (untreated) conditions. In contrast, in RACT-treated R21 Rex1−/− cells, the Src DN mutant reduced the SOCS-3 promoter/reporter activity by 2.79 ± 0.22 fold (n=3, p=0.023) and the Jak2 DN mutant reduced SOCS-3 promoter/reporter activity by 3.21 ± 0.27 fold (n=3, p=0.034). The apparent reduction of SOCS-3 promoter/reporter activity by the Jak1 DN mutant in RACT-treated R21 cells was not statistically significant (p>0.05). We conclude that Src and Jak2 are involved in the enhanced activation of SOCS-3 transcription by RACT treatment in Rex1−/− cells.
To assess whether the RA:RAR complex played a role in the activation of SOCS3 transcription we transiently co-transfected the murine RARα in an expression vector into F9 Wt vs. Rex1−/− R21 cells, and then cultured the cells ± RACT for 48 hr. pSG/RARα transfection increased the activity of the SOCS3 promoter (construct 6) by ~2-fold over the activity with empty pSG5 vector co-transfected in both the F9 Wt and the R21 mutant. This increase did not take place when the SOCS3 promoter construct 6D2C, which lacks the STAT3 binding sites, was co-transfected (Fig. 9). Thus, in the presence of RA RARα can stimulate SOCS3 promoter activity, but this stimulation requires the STAT3 binding sites.
To examine the effect of PTH on RA-induced SOCS-3 mRNA in F9 cells, PTH was added to the cells at the same time that RA was added. As a control, PTH peptides were added to the cells alone. In F9 Wt cells, the addition of PTH alone did not have any effect on SOCS-3 mRNA levels. When PTH was used in combination with RA, PTH peptide did not enhance the action of RA on SOCS-3 mRNA expression in F9 Wt cells. In control F9 Rex1−/− (R21) cells, the addition of PTH alone did not have any effect on SOCS-3 transcript level. In contrast, the combination of PTH and RA greatly increased SOCS-3 mRNA levels in R21 Rex1−/− cells (Fig. 10A).
BME is a membrane permeant reducing agent which has been shown to inhibit the actions of reactive oxygen species (ROS), and thereby, inhibit activation of the JAK/Stat pathway75; 79; 80. To examine the effect of BME on RACT-induced SOCS-3 transcript levels, BME was added to the cells at the same time RACT was added. In both F9 Wt and F9 Rex1−/− cells, the addition of BME partially blocked the effect of RACT on SOCS-3 mRNA expression (Fig. 10A). These results suggest that ROS is involved in RACT-induced SOCS-3 mRNA expression though tyrosine phosphorylation.
In the absence of Rex1, the transcriptional activation of the SOCS-3 gene by RA, CT, and RACT was greatly augmented (Figure 4). Using transient transfection of promoter/reporter constructs, we showed that the STAT3 tandem binding sites, located between −99 and −60 in the 5' genomic region, were required for the high activity of the SOCS-3 promoter/reporter in the F9 Rex1−/− cells, but not in the F9 WT cells (statistically not significant) (Figure 4B). Deletion of the STAT3 binding sites in the SOCS-3 promoter (construct 6D2C) lowered the activity of the SOCS-3 promoter/reporter activity by ~6 to ~8-fold in F9 Rex1−/− cells (Figure 4B).
The two STAT3 DN mutants, Y705F and D715, also reduced SOCS-3 promoter (construct 6, Fig. 4A, B) activity in Rex1−/− cells to a level that was similar to the activity of construct 6D2C (Figure 5B). The reduction by the STAT3 DN mutant Y705 was greater than that seen with the STAT3 DN mutant D715. These data also indicate that STAT3 activation is required for the high level of transcriptional activation of the SOCS-3 gene in F9 Rex1−/−cells after CT or RACT treatment.
The co-transfection of the constitutively active mutant of STAT3 (STAT3C) activated the SOCS-3 promoter by two-fold in F9 Wt cells; this most likely results from a greater number of STAT3 dimers bound to the SOCS-3 regulatory region (Fig. 5). A similar twofold increase in the presence of STAT3C was observed in RA, CT, and RACT-treated F9 Wt and F9 Rex1−/−cells (Figure 5B). In F9 Rex1−/−cells RA alone had an effect similar to CT alone or RACT on SOCS-3 promoter6/reporter activity (Fig. 5B, see STAT3C, RA treatment).
According to the negative autoregulatory circuit proposed for STAT3/SOCS-3, tyrosine phosphorylated STAT3 activates SOCS-3 transcription. Subsequently, SOCS-3 protein inhibits the activation of STAT337; 81; 82. We report here that in F9 Rex1−/− cells, the levels of both phosphorylated STAT3 and SOCS-3 greatly increase in response to RACT treatment (Figs. 1, ,2,2, ,3).3). This is in contrast to the reduction in phosphorylated STAT3 that would generally be expected with increased SOCS-3 protein.
To test the involvement of PKA in SOCS-3 transcriptional activation, we co-transfected an expression construct containing a PKA DN mutant with the SOCS-3 promoter/reporter. The PKA DN mutant did not have any effect on control or RA-treated cells (both Wt and Rex1−/− cells) (Figure 7). In contrast, the transfection of the PKA DN mutant lowered the SOCS-3 promoter/reporter activity by ~3.5 fold in CT and RACT-treated F9 Wt and F9 Rex1−/− cells (Fig. 7). Similarly, Bousquest et al.41; 83 reported that in addition to cytokines, cAMP agonists, alone or with LIF, increased SOCS-3 promoter activity in AtT20 cells. A consistent, additive effect was seen between cAMP analogs and LIF, and the cAMP actions were shown to be mediated via PKA. A dominant negative PKA reduced the cAMP mediated induction of the SOCS-3 promoter by 50%83. Bousquet et al.41; 83 reported that cAMP activated SOCS-3 transcription in AtT20 cells via an AP-1 site (TGACTAA) at −105/−99. C-fos and Jun B, but not phospho-CREB, bound to this site in gel shift assays41. The AP-1 site and the STAT3 sites are very close together in the SOCS-3 promoter. However, we have shown that the enormous activation of the SOCS-3 promoter in the Rex1−/− cells is completely dependent on the STAT-3 sites in the promoter (Figs. 4 and and5).5). In addition, a dominant negative c-jun expression vector, transfected with the SOCS3(6) promoter into RACT treated Rex1−/− cells, did not decrease the promoter activity (data not shown). Thus, our data are more consistent with the effect of cyclic AMP in the Rex1−/− cells being exerted through an increased level of tyrosine phosphorylated STAT-3 protein (Fig. 6B) rather than through c-fos, junB, or CREB binding at a separate site on the SOCS-3 promoter.
Another mechanism by which cyclic AMP could enhance SOCS-3 transcription in the presence of RA is via phosphorylation of the activation functions of the RARs by pkA84; 85. Additional mechanisms of crosstalk between the RA and the STAT3 signaling pathways have been reported in leukemia cells31; 86; 87.
STAT proteins can be activated by a variety of receptor and non-receptor protein tyrosine kinases71; 72; 73; 75; 76; 86; 88; 89. To delineate the pathway that activates STAT3 in RACT-treated F9 cells, we further explored the signaling pathway upstream of STAT3. First, we examined the effects of Src DN, Jak1 DN, and Jak2 DN mutant expression constructs on SOCS-3 promoter/reporter activity (Fig. 8). The transfection of each of these mutants did not significantly alter the SOCS-3 promoter/reporter activity in F9 WT cells (Figure 8B). In RACT-treated F9 Rex1−/− cells, the transfection of either the Jak2 DN or Src DN lowered the SOCS-3 promoter/reporter activity by ~3-fold. However, neither the Src DN expression construct nor the Jak2 DN construct had as potent an effect as the STAT3 DN mutants (Fig. 5) on the SOCS-3 promoter/reporter activities. A dominant negative MEK expression vector, transfected into RACT treated F9 Rex1−/− cells with the SOCS3(6) promoter vector, did not cause any decrease in SOCS3 promoter activity (data not shown). This indicates that the MEK pathway is not required for the enhanced SOCS3 promoter activity seen in the Rex1 null cells.
The classical pathway of STAT3 activation is through LIF/LIFR/gp130/Jak290; 91. However, there are several pieces of evidence that LIF is not involved in the enhanced STAT3 activation in F9 Rex1−/− cells. First, LIF is not required for F9 cells to remain undifferentiated92, and LIF is not present in the culture medium. Second, the LIF pathway is rapid, e.g. only thirty minutes are required for STAT3 phosphorylation following LIF addition 93. Third, LIFR levels are similar in F9 WT cells and F9 Rex1−/− cells (Tighe and Gudas, unpublished data).
It has been reported that the simulation of the G-protein coupled receptor (GPCR) family members leads to tyrosine phosphorylation of JAKs and STATs, and subsequently induces STAT binding activity in target cells94. Pelletier et al.94 reported that the GPCR agonists thrombin and angiotensin II could stimulate tyrosine phosphorylation of STAT3 in a Rac-dependent manner in smooth muscle cells. They provided solid data suggesting that the late, second phase of STAT3 activation by GPCRs depends on the autocrine production of a ligand, possibly IL-6, by a Rac-dependent mechanism. Pettelier et al. suggested that GPCR activation leads to Rho activation, and subsequently, reactive oxygen species (ROS) are activated, resulting in the activation of the Jak2/STAT3 pathway94. PTHR (parathyroid hormone receptor) is a member of the GPCR family95 and the expression of PTHR mRNA is higher in F9 Rex1−/− cells than in F9 WT cells after RA treatment (Fig. 10A). It was reported that PTH (parathyroid hormone) activates Src kinase activity96 and Src kinase activates Rho family members by phosphorylation97. Thus, PTHR may be involved in the activation of Jak2 and STAT3 in RACT treated F9 Rex1−/− cells (Fig. 10B).
In RACT treated F9 Rex1−/− cells phosphorylated STAT3 levels are very high (Fig. 6), indicating that the Rex1 protein plays an inhibitory role in this pathway in F9 cells. In the absence of Rex1 and with RACT treatment, then, higher levels of expression of receptors such as PTHR (Fig. 10A, B) would lead to increased tyrosine phosphorylation and activation of STAT3. This increase in activated STAT-3 would then result in the increased transcription of the SOCS-3 gene observed in the RACT treated Rex1−/− cells. We found no functional evidence of Rex1 directly binding to the SOCS-3 promoter, i.e. a promoter element, which when deleted, would elevate the level of SOCS-3 promoter driven luciferase activity (Xu and Gudas, unpublished). Thus, our data suggest an indirect role for Rex1 protein in this pathway, possibly, for example, via stimulating the transcription of an inhibitor of STAT3 such as GRIM1998; 99; 100; 101 (Fig. 10B). This model is consistent with our experimental data which indicate that the enhanced levels of SOCS-3 mRNA in Rex1 null cells occur at later times (>48 hr.) after RA addition (Figs. 1, ,22).
F9 WT cells, two homozygous knockout lines [Rex1−/−-2-1 (R21), Rex1−/−−5 (R5)]22, and an exogenous Rex1 expression “put back” line (R21JXR12) were maintained in DMEM (Dulbecco's Modified Eagles Medium) containing 10% heat-activated calf serum and 2 mM glutamine. To culture cells in monolayer, the flasks and dishes were treated with 0.3% gelatin before plating the cells. To generate primitive endoderm (PrE), F9 cells were grown in monolayer culture in the above medium containing 1μM all-trans RA (Cat# R-2625, Sigma, St. Louis, MO). Medium on the cells was replaced every 48 hours with fresh medium containing 1μM RA. To generate parietal endoderm (PE), F9 cells were grown in the above medium containing 1μM RA, 250μM dibutyryl cyclic AMP (db cAMP) (N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, Sigma Cat# D-0627), and 250 μM theophylline (Sigma Cat# T-1633). Medium on the cells was replaced every 48 hours with fresh medium containing RA, db cAMP, and theophylline (RACT). PTH 1–34 was from Sigma (#P4056).
F9 WT cells and F9 Rex1−/− cells were either untreated or treated with 1 μM RA for 48 hours, and total RNA was then extracted. Microarray analysis was carried out according to the Affymetrix Genechip expression analysis technical manual. Briefly, total RNA from untreated and RA-treated (1 μM RA for 48 h) F9 WT and F9 Rex1−/− cells was reverse transcribed into cDNA. After second-strand synthesis, cDNAs were then in vitro transcribed into cRNAs with biotinylated ribonucleotides (Enzo Diagnostics, Farmingdale, NY). cRNA (20 μg) was fragmented by heating at 94 °C for 35 minutes. A cocktail containing fragmented cRNA, control oligonucleotide B2, control cRNA (Biotin B, Biotin C, Biotin D, and Cre), and herring sperm DNA was hybridized to microarray chips (MG-U74Av2, 11,938 mouse genes) for 16 hours at 45 °C. After washing and staining, the distribution of fluorescent material on the array was measured using a laser scanner. The resultant image was processed with MAS (Microarray Suite) software (Affymetrix, Santa Clara, CA). The microarray experiments were repeated two times with different RNA preparations. Only genes which exhibited reproducible differences in expression patterns among the four groups of cells were further analyzed.
The Microarray Core Facility of Weill Medical College of Cornell University performed the data analysis. The expression level of each gene and the fold change in expression in untreated versus RA-treated F9 Wt cells and F9 Rex1−/− (R21) cells were calculated using a computer program (GeneSpring, Redwood, CA). This computer program was also used for functional gene grouping analyses (not shown).
F9 Rex1−/−-2-1 cells (4 × 106 cells/150 μm plate were set up the night before) were co-transfected with 40 μg of pREX-SG plasmid and 4 μg pSV2Neo plasmid by the calcium phosphate precipitation method102. This pREX-SG plasmid was constructed by Dr. Betsy Hosler103. pREX-SG contains the full length mouse Rex1 cDNA driven by the SV40 promoter; the Rex1 full length cDNA is in the correct orientation for expression of the murine Rex1 protein. Both plasmids were prepared using the QIAGEN EndoFree™ Maxi Kit (QIAGEN Inc., Valencia, CA), and supercoiled DNAs were used for the transfection. Three hours prior to transfection, the old medium was replaced with 20 ml of fresh medium. For the transfection, DNA was suspended in 840 μl 1mM Tris (pH 7.4) first; then, 120 μl of 2M CaCl2 was added dropwise with agitation to the DNA; finally, the DNA-CaCl2 was added to 960 μl 2× HBS (pH=7.05) with bubbling, the reaction (Tris + DNA + CaCl2 + 2× HBS) was incubated for 20 minutes at room temperature, and then added dropwise to the cells. The precipitates were removed after 16 hours, and then the cells were washed with 1× TBS. Approximately 48 hours after transfection, G418 (Gibco, Carlsbad, CA, Cat#11811-0311) was added to a final concentration of 390 μg/ml active drug. The cells were cultured in medium with 300 μg/ml active G418 for three weeks, and 70 G418 resistant cell colonies were picked for analysis. Each resistant colony was individually expanded, a duplicate well was frozen, and total RNA was extracted for Northern analyses to assay for the Rex1 positive clones.
Cells were either untreated, treated with RA, CT, or RACT for 6 hr, 24 hr, 48 hr, or 72 hr. After 48 hours, medium was changed and fresh drugs were added. Cell monolayers were washed twice with ice-cold PBS and harvested in 200 μl 2X SDS-PAGE buffer buffer (240 mM Tris-HCl, pH6.8, 8% SDS (sodium dodecylsulfate), 40% glycerol, without bromphenol blue). Cell lysates were then passed through 26G5/8 needles (VWR, West Chester, PA, #309597). Then, cell lysates were boiled for 5 minutes, and cell debris were removed by centrifuging the cell lysates for 30 seconds in a microfuge at maximum speed in the cold. Some lysates were used for protein concentration measurements using the DC Protein Assay (Bio-Rad, Hercules, CA, #500-0114). Dithiothreitol was added to the remainder of the cell lysates to a final concentration of 100 μM, and lysates were stored at −20 °C or at −70°C. Then, 45 μg (for STAT3 analysis) or 100 μg (for SOCS-3 analysis) of total cell extract was mixed 1:1 with 2X SDS-PAGE buffer with 0.1% bromphenol blue, boiled for 5 minutes, and separated on a 7.5% SDS-PAGE gel for STAT3 and a 12% gel for SOCS-3. The gel was electrophoretically transferred to a nitrocellulose membrane (Bio-Rad, #162-0090). The membrane was blocked in 5% BLOTTO (Santa Cruz Biotechnology, Santa Cruz, CA, #sc-2325) made in PBS-0.5% Tween 20 for 3 hours at room temperature. Then, the membranes were incubated with primary antibodies to STAT3 (Upstate Biotechnology, Inc., Charlottesville, VA, #06-596) 1:2,000; Phospho-STAT3 (Cell Signaling Technology, Beverly, MA, #9131) 1:2,000; SOCS-3 (Santa Cruz Biotechnology, #sc-7009) 1:200; and actin (Santa Cruz Biotechnology, #sc-1616) 1:2,000, overnight at 4°C, and with secondary antibodies: anti-rabbit IgG-horseradish peroxide (HRP) (Santa Cruz Biotechnology, #sc-2030) 1:5,000; or anti-goat IgG-HRP (Santa Cruz Biotechnology, #sc-2056) 1:7000 for 2 hours at room temperature. Results were visualized by enhanced chemiluminescence reaction using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL, #34080), according to the manufacturer. To detect the luminescent signal, blots were exposed to film and developed.
Northern analysis was performed as previously described104. Total RNA was prepared from cells using the RNA-BEE™ (TEL-TEST ”B”, Inc., Friendswood, TX, #CS-104B). Each RNA sample (10 μg) was run on a 6.2% formaldehyde agarose gel. RNA was transferred to a Hybond-N nylon membrane (Amersham Biosciences, Piscataway, NJ, #RPN303) and hybridized to individual probes in standard hybridization solution containing 5X SSC, 5 mM EDTA, 10X Denhardts, 0.1% SDS, 10% dextran sulfate, 100 μg/ml Salmon Sperm DNA, and 50% formamide. Probes were radiolabeled using the Random Primed DNA Labeling Kit (Roche Diagnostics, Indianapolis, IN, #1004760). Hybridized filters were washed two times under low stringency conditions (0.1% SDS and 2X SSC) at 42 °C for 20 minutes each and then were washed once under high stringency (0.1% SDS and 0.2X SSC) at 60 °C for another 20 minutes. After washing, the filters were exposed to Kodak BioMax film, and a Phosphorimager. Each filter was also hybridized to GAPDH as a loading control. We obtained a cDNA for murine SOCS-3 from Dr. J. Ihle at St. Jude Children's Research Hospital, and sequenced this to confirm that it was SOCS-3.
PCR was performed in a total volume of 20 μl using 10 ng of the template (clone 6), 10 pmol each of either the forward or reverse primer, 200μmol dNTP, 1X Cloned Pfu DNA polymerase reaction buffer (Stratagene, La Jolla, CA, Cat# 600250), and 2.5 U PfuTurbo DNA Polymerase (Stratagene, Cat# 600250). PCR reactions were carried out in a PTC-100 Peltier Thermal Cycler (MJ Research, Waltham, MA) for 30 cycles with the following parameters: 94 °C for 45 seconds, 55 °C for 45 seconds, 72 °C for various lengths of time (1 minute per kb). Before the 30 cycles, PCR reactions were heated at 94 °C for 5 minutes to separate double-stranded DNA. After 30 cycles, PCR reactions were extended for another 10 minutes at 72 °C for final nucleotide extensions.
Murine SOCS-3 promoter clones 2, 6, and 6D2C were kindly provided by Dr. S. Melmed from Cedars-Sinai Research Institute. All three of these constructs were cloned into the pGL3-Basic vector by Auernhammer et al42; 83. Clone 6 contains the −2757 to +929 5' genomic region of murine SOCS-3. Clone 2 is a truncation of clone 6 (nucleotides −2757 to −714) and lacks the minimal promoter region. In clone 6D2C (referred to as 6D2 by Auernhammer et al.42), the complete tandem STAT binding region from nucleotides −99 to −60 was deleted42.
All of the inserts for the SOCS-3 promoter deletions and mutations were obtained by PCR. PCR products were run on a 1% agarose gel and the PCR products were gel-purified by QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, Cat# 28706). Purified PCR products and either the pGL3-Basic vector (Promega, Madison, WI, lacks eukaryotic promoter and enhancer sequences) or pGL3-Promoter vector (Promega, contains SV40 promoter located upstream of the luciferase gene) were cut by designated restriction enzymes overnight. Then, restriction digested PCR products and vectors were run on a 1% agarose gel and gel-purified. The gel-purified PCR products and vectors were ligated using the Rapid DNA Ligation Kit (Roche Diagnostics, Cat# 1635379). The ligated products were transformed into the host strain, DH5α. The products were confirmed by DNA sequencing.
1.5 × 106 F9Wt cells/100mm plate and 1.3 × 106 F9 Rex1−/− cells/100 mm plate were plated the night before transfection. Three hours before transfection, the medium was changed. Then, cells were transiently transfected with 4 μg of reporter plasmid and 1.5 μg of Renilla luciferase plasmid (Promega) by the calcium phosphate precipitation method. Plasmids were prepared using the QIAGEN EndoFree™ Maxi Kit (QIAGEN Inc., Valencia, CA), and supercoiled DNAs were used for the transfection. For the transfections: first, DNA was brought up to 420 μl in 1 mM Tris (pH 7.4); then, 60 μl of 2M CaCl2 were added dropwise with agitation to DNA; finally, DNA-CaCl2 was added to 480 μl 2XHBS with bubbling, the reaction was incubated for 20 minutes at room temperature, and then was added dropwise to the cells. The precipitates were removed after 16 to 18 hours and then the cells were washed with 1X TBS. Then, RA, CT, and RACT were added with fresh medium immediately after washing. Forty-eight hours after drug treatments, cells were harvested for dual-luciferase assays.
To study the effects of the retinoic acid receptors (RARs), expression vectors containing the full-length murine RARα or RARγ downstream of the SV40 promoter were transiently co-transfected. In some experiments an expression plasmid with the full-length murine Rex1 cDNA downstream of the SV40 promoter was co-transfected. To study the effects of these various constructs: WTSTAT3, Y705F (STAT3 dominant negative (DN) mutant), D715 (STAT3 DN mutant), STAT3-C (constitutively active mutant of STAT3), a Jak1 DN mutant, a Jak2 DN mutant, and a Src DN mutant, 4 μg of each of the above constructs was cotransfected with 1.5 μg of the Renilla luciferase construct and 4 μg of clone 6 of the SOCS-3 promoter. For a negative control, 4 μg of the pUC9 vector was co-transfected with 1.5 μg of the Renilla luciferase construct and 4 μg of clone 6 of the SOCS-3 promoter. To study the effect of MT-REV(AB) (protein kinase A DN) on SOCS-3 promoter activity, 3 μg of the MT-REV(AB) construct was co-transfected with 1.5 μg of the Renilla luciferase construct and 4 μg of clone 6 of the SOCS-3 promoter. For the negative control, MT64AA [the MT-REV(AB) empty expression vector] was co-transfected with 1.5 μg of the Renilla luciferase construct and 4 μg of clone 6 of the SOCS-3 promoter. To induce the expression of MT-REV (AB), ZnCl2 was added to the cells to a final concentration of 50 μM at the same time that RA, CT, and RACT were added.
Fourty-eight hours after drug treatments, transiently transfected cells were harvested in 1X Passive Lysis Buffer (Promega, Cat# E194A). Cell lysates were first stored at −20 °C overnight. Before luciferase assays, cell lysates were thawed and the cell debris were removed by centrifuging at maximum speed in a microfuge for 30 seconds at room temperature. For F9 Wt and Rex1−/− samples 20 μl (~5 μg) of cell lysates were used to perform luciferase assays according to the manufacturer's protocol for Dual-Luciferase® Assay System (Promega, Cat# E1960). The amounts of cell lysates used for the luciferase assays were determined by running concentration curves and the amounts used were within the linear ranges for luciferase activity/μg protein. For each assay, the luminometer was programmed to provide a 2-second preread delay, followed by a 10-second measurement period. The luciferase values were recorded by a TD-20/20 luminometer (Turner Designs, Inc., Sunnyvale, CA).
We would like to thank the following investigators for their generous gifts: Dr. James Ihle for SOCS-3 cDNA used for Northern Analyses; Dr. Shlomo Melmed for the SOCS-3 promoter clones 2, 6, and 6D2C; Dr. Chris Auernhammer for discussions about the SOCS-3 promoter constructs; Dr. Shizuo Akira for the STAT3 WT construct and the STAT3 DN mutants; Dr. David Levy for the JAK DN mutants; Dr. G. Stanley McKnight for the PKA DN mutant, and Dr. Jacqueline Bromberg for the STAT3C construct. We thank Karl B. Ecklund for editorial assistance. This work was supported by NIH grants to LJG, and in part by the U. S. Army Medical Research, award number DAMD 17-01-1-0218 to JX.
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