A mutant cell line partially responsive to both IFN-<FONT FACE=Symbol>a</font> and IFN-<FONT FACE=Symbol>g</font>

Abstract

A recessive mutant cell line, B7, which is partially responsive to both interferon (IFN)-<FONT FACE="Symbol"> a</font> and IFN-<FONT FACE="Symbol">g</font> is described. B7 was FACS sorted from a cellular pool, which was obtained from the parental cell line 2C4, after several rounds of mutagenesis. The partial responsiveness to IFN was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (9-27; 6-16; 2'-5' OAS; GBP and HLA-DRa). A genetic cross with the U4 mutant (JAK1-, a member of the Janus family of nonreceptor tyrosine kinase) did not restore full IFN-responsiveness to B7, and JAK1 cDNA transfection into B7 restored the wild phenotype of the cell line, defining B7 as a member of the U4 complementation group. Nevertheless, JAK1 mRNA was not detected in this mutant. Transcriptional regulator complexes such as IRF1/2 (IFN-regulatory factor) and ISGF3-<FONT FACE="Symbol">g</font> (IFN-stimulated gene factor) were constitutively formed in the B7 mutant and co-migrated with the IFN-induced complexes expressed in the parental cell line 2C4. Thus, this cell line seems to be useful for understanding cis-acting elements governing JAK1 mRNA expression.

interferons; transcriptional activation; molecular genetics; JAK1; FACS analysis

Braz J Med Biol Res, January 1997, Volume 30(1) 41-50

A mutant cell line partially responsive to both IFN- a and IFN- g

C.A. Bonjardim

Laboratório de Vírus, Departamento de Microbiologia, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brasil

^{Correspondence and Footnotes}

Abstract

A recessive mutant cell line, B7, which is partially responsive to both interferon (IFN)-a and IFN-g is described. B7 was FACS sorted from a cellular pool, which was obtained from the parental cell line 2C4, after several rounds of mutagenesis. The partial responsiveness to IFN was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (9-27; 6-16; 2’-5’ OAS; GBP and HLA-DRa). A genetic cross with the U4 mutant (JAK1^-, a member of the Janus family of nonreceptor tyrosine kinase) did not restore full IFN-responsiveness to B7, and JAK1 cDNA transfection into B7 restored the wild phenotype of the cell line, defining B7 as a member of the U4 complementation group. Nevertheless, JAK1 mRNA was not detected in this mutant. Transcriptional regulator complexes such as IRF1/2 (IFN-regulatory factor) and ISGF3-g (IFN-stimulated gene factor) were constitutively formed in the B7 mutant and co-migrated with the IFN-induced complexes expressed in the parental cell line 2C4. Thus, this cell line seems to be useful for understanding cis-acting elements governing JAK1 mRNA expression.

Key words: interferons, transcriptional activation, molecular genetics, JAK1, FACS analysis

Introduction

Interferons (IFNs) constitute a family of multifunctional cytokines first identified as antiviral agents. However, other activities have been associated with these molecules such as the control of cell growth and differentiation, besides their modulatory regulation of the immune system (1).

After binding to the specific cell surface receptor, type I IFN (mainly a and ß) and type II IFN (g) activate a number of IFN-stimulated genes (ISGs), which are believed to be responsible for the different IFN actions (1,2). IFN-activated gene expression is mediated by latent cytoplasmic proteins which become phosphorylated at a tyrosine residue upon ligand-receptor binding, migrate to the nucleus, bind to DNA and initiate transcription (3-5).

It has been shown that the receptor to nucleus signalling triggered by IFN-a relies on the activation of the primary transcriptional activator IFN-stimulated gene factor 3 (ISGF3), which comprises the a and g sub-units (6,7). The ISGF3-a moiety is constituted of three polypeptides: 113 kDa (p113), 91 kDa (p91) and 84 kDa (p84). p91 and p84 are products of a differentially spliced gene (8-10) and the ISGF3-g moiety is represented by p48, which is the DNA-binding protein (9). The ISGF3-a and -g subunits associate after tyrosine phosphorylation of the a-subunit proteins, which in turn translocate to the nucleus, bind to cis-acting DNA elements termed interferon-stimulated response element (ISRE) located at the promoter of ISGs and initiate transcription (11-13). Due to the dual role of p91, p84 and p113 as signal transducers and activators of transcription, these molecules were renamed STAT1a, STAT1ß and STAT2, respectively.

The nuclear signals resulting from the interaction of IFN-g with its receptor occur in a fashion very similar to that described for IFN-a. After activation of STAT1a (the same component of ISGF3-a), and migration to the nucleus, STAT1a activates transcription by binding as a dimer (14) to a regulatory sequence termed gamma-activated sequence (GAS). GAS was initially identified at the promoter of the guanylate-binding protein (GBP) gene (15,16), but has now been found in a variety of other genes (17-19), and STAT1a turned out to be the previously described gamma-activated factor (GAF) (20).

It is known that the intracytoplasmic portions of both IFN-a/ß and IFN-g receptors are devoid of an intrinsic tyrosine kinase activity (21,22), suggesting that a nonreceptor protein tyrosine kinase (NRPTK) is recruited to assemble the multisubunit receptor complexes in order to transduce the nuclear signals (23,24). By using a genetic approach, a number of IFN-unresponsive cell lines (U- and g-mutants) have been generated (25-28) and genetic complementation of these mutants by cDNA transfection has proved valuable in dissecting both types of IFN signalling pathways (29-31).

The first evidence that a NRPTK is involved in these pathways came from the complementation of the U1 mutant which was transfected with the gene and cDNA encoding for TYK2 (29), a member of the Janus family of NRPTK (23,24). Complementation of the U3 mutant, a STAT1a^-/STAT1ß^- cell line, established the fundamental role STAT1a plays in the IFN-a and IFN-g transduction pathways (30), while complementation of the U4 mutant, a JAK1^- (a member of the Janus family of NRPTK) cell line, clearly demonstrated the involvement of NRPTK-JAK1 in both signalling pathways (32). Another important finding was the restoration of the IFN-g responsiveness to the JAK2^- cell line (g1A) upon JAK2 cDNA transfection (28). Thus, the converging biochemical and genetic approaches enable to decipher the emerging picture of both IFN-a and IFN-g signalling cascades (5), which can be summarized as follows: IFN-a: two JAK family members are required: TYK2 and JAK1, plus the transcription factors p113 and/or STAT1a/STAT1ß, besides p48. IFN-g: two JAK family members are required as well: JAK1 and JAK2 , plus the transcription activator STAT1a (dimer), probably associated with a DNA-binding protein.

In the present study, I describe the genetic characterization of a mutant cell line, B7, and its complementation by JAK1 cDNA. Although JAK1 mRNA expression was below detection levels in B7, a partial IFN-a and IFN-g responsiveness was achieved. In addition, DNA-protein complexes are constitutively formed in this mutant. Thus, this cell line represents a useful model to analyze JAK1 structure-function relationships, concerning JAK1 expression.

Material and Methods

Cell culture, cell fusion and DNA transfection

Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 5 µM L-glutamine, and neomycin (G418, 700 µg/ml) and incubated at 37^oC, 5% CO₂. The parental cell line 2C4 was generated as described elsewhere (28). Briefly, human HT1080 cells were co-transfected with a selectable marker, pTKNeo, and pDW9-27CD2, a plasmid containing the promoter region of the IFN-stimulated gene 9-27 placed just upstream of a full-length CD2 cDNA. 2C4 cells were mutagenized (five rounds) with the frameshift agent ICR191 (25) and the cell population was subdivided into different pools. B7 was FACS sorted from one of these pools (a gift from D. Watling, ICRF, London), cloned and characterized. U3 and U4 mutants were characterized as described elsewere (27), and both are unresponsive to type I and type II IFN and correspond to STAT1a^-/1ß^- and JAK1^- cell lines, respectively. For cell fusion experiments a puromycin- or hygromycin-dominant selectable marker was transfected into the cells and the resistant hybrid cell population was maintained in the presence of the adequate drug selection. Cell fusion experiments employing PEG 4000 (Sigma) were performed as described elsewhere (27). DNA transfections using calcium phosphate were carried out essentially as described in Ref. 30.

FACS analysis

Fluorescence-activated cell sorter (FACSCAN-Becton Dickinson) analysis was performed as previously described (28). Briefly, 5 x 10⁵ cells were seeded in a 10-cm Petri dish and after an overnight incubation they were treated with 10³ IU/ml of a highly purified mixture of IFNs-a (Wellferon, 1.5 x 10⁸ IU per mg protein) from Wellcome Research Laboratories, Beckenham, Kent, UK, or recombinant human IFN-g (2 x 10⁷ IU per mg protein), a generous gift from Dr. G. Adolf, Ernst Boehringer Institut für Arzneimittelforschung, Vienna, Austria. Cells were treated with IFN for 48-72 h and then incubated with antibodies as described in Ref. 28 for 1 h at 0^oC. Cells were pelleted, resuspended in PBS, fixed in 1% paraformaldehyde and analyzed in a FACS using a Consort 30 program (3000 data points).

RNase protection assay

Cytoplasmic RNA was obtained from monolayer cells by NP40 lysis and phenol/chloroform extraction. Probes were synthetized from SP6/T7 transcription vectors, pGEMs 3 and 4 (Promega), labelled with [³²P]aUTP (Amersham, UK) to a specific activity >10⁸ cpm/µg DNA. Ten µg of RNA and 1-3 x 10⁵ cpm of each probe were employed in each assay. Single-stranded RNA was digested with ribonucleases and protected double-stranded RNA was resolved on a 6% polyacrylamide-urea gel as described elsewhere (30). Details on the construction and length of the protected fragments of the specific RNase protection probes (2’-5’ OAS; 6-16; GBP; HLA class II DRa; 9-27 and g-actin) used in this work have been described elsewhere (30). The 6-16 and GBP probes were gifts from S. Goodbourn and T. Decker, respectively.

Electrophoretic mobility shift assay (EMSA)

EMSA was carried out essentially as described in Ref. 30. Whole cell extracts were prepared by a modification of the method of Zimarino and Wu (33). Briefly, frozen-cell pellets were thawed on ice and lysed with an equal volume of extraction buffer (0.76 M NaCl, 1.5 mM MgCl₂, 10 mM HEPES, pH 7.9, 0.1 M EGTA, 5% glycerol, 0.5 mM dithiothreitol, and 0.2 mM phenylmethyl-sulfonylfluoride). Lysates were centrifuged at 100,000 g at 4^oC for 30 min. The supernatants were dialyzed against the same buffer as described above, except that 0.1 M NaCl was used, for approximately 4 h at 4^oC, and the concentration of protein was determined by the Bio-Rad assay. Ten µg of protein was pre-incubated with 1.2 µl poly dI-dC (Pharmacia) (5.4 mg/ml) at room temperature for 10 min, followed by the addition of 1.25 µg BSA, 0.125 µg E. coli DNA, 0.25 µg yeast tRNA, 2% Ficoll 400 and 0.32 ng labelled probe. The reactions were incubated at room temperature for 15 min and then analyzed by 6% PAGE. The 5’ [³²P]-end-labelled probes 6-16, 9-27 and IRF-1 were used as described elsewhere (34,35).

Northern blot analysis

mRNA poly A⁺ was obtained according to the manufacturer (Pharmacia). Five µg RNA per lane was electrophoresed on a denaturing 1.5% agarose-formaldehyde gel, transferred to Hybond N⁺ membranes (Amersham, UK) as previously described (36) and hybridized with an adCTP [³²P] human JAK1 cDNA probe, labelled to a specific activity >10⁸ cpm/µg DNA, by using a multiprime DNA labelling system from Amersham, UK. Hybridization was performed at 65^oC and subsequent washes were carried out according to Church and Gilbert (37). After exposure for 7 days, the membrane was stripped of the JAK1 probe and then reprobed with GAPDH, labelled at the same specific activity as described above, and exposed for 16 h.

Results

FACS analysis of the parental cell line 2C4 and the B7 mutant

FACS analysis of the B7 mutant indicated that this cell line responded partially to IFN-a and IFN-g when the cell surface marker (CD2) and the endogenous class I HLA were investigated, whereas no response to IFN-g was observed when the expression of endogenous class II HLA was analyzed (Figure 1A, panels 4-6). The expression of these genes in response to both IFN-a and IFN-g in the parental cell line 2C4 is shown in Figure 1A (panels 1-3). B7 is a recessive mutant since crossing with 2C4 and with the wild type 2fTGH (25) restores the full responsiveness of the cells to both types of IFN (data not shown). The mutation(s) in B7 seems to be very stable since its phenotype remained the same after several passages in culture for more than three months, as confirmed by FACS analysis and RNase protection experiments.

Figure 1
- FACS analysis of the parental and mutant cell lines. A, 2C4, B7 and B7/JAK1 transfectants and B, genetic crosses B7/U3 and B7/U4. The expression of the cell surface markers CD2 (panels 2,5,8,11,14) and endogenous class I (panels 1,4,7,10,13) and class II HLAs (panels 3,6,9,12,15) upon IFN-a or IFN-g stimulation (500 IU/ml for 48 h) was monitored by FACS analysis as described in Material and Methods. Transfectants were analyzed as a population. Panels 1-3: Parental cell line 2C4; panels 4-6: B7 cell line; panels 7-9: B7/JAK1 transfectants; panels 10-12: genetic cross between B7 and the U3 mutant; panels 13-15: genetic cross between B7 and the U4 mutant. Untreated cells (U), IFN-a (a) or IFN-g (g) treated cells, and unstained cells (US) as indicated. US cells were left untreated or IFN-a treated, but were not incubated with any antibody.

JAK1 complements B7 mutant

From the FACS analysis data (Figure 1A, panels 4-6), it was evident that the nature of the mutation(s) affecting both IFN-a and IFN-g signalling pathways in this particular cell line may reside in a common polypeptide(s) which serves simultaneously in both the IFN-a and IFN-g transduction cascades. To address this question, the mutant cell lines U3 and U4, which are known to be unresponsive to both types of IFN (27), were fused to the B7 mutant and cell surface expression of CD2, and class I and II HLAs were monitored by FACS analysis as described elsewhere (28). Full responsiveness to both IFN-a and IFN-g was obtained after fusing B7 with the U3 mutant (Figure 1B, panels 10-12). However, fusion with U4 just maintains the same original responsiveness of B7 to both types of interferon (Figure 1B, panels 13-15), indicating that B7 is a member of the U4 complementation group. Human JAK1 cDNA was then transfected into the B7 cell line, and cell surface expression (the same markers as described above) was monitored by FACS analysis (Figure 1A, panels 7-9). Although the experiment was done with a cell population, an enhancement of responsiveness to both IFN-a and IFN-g was achieved, confirming B7 as a member of the U4 complementation group.

IFN-inducible gene expression

I next asked whether the IFN-a- and IFN-g-partial responsiveness observed in this cell line by FACS analysis is correlated with the IFN-a- and IFN-g-gene inducibility. Figure 2 shows mRNA expression of some IFN-stimulated genes (2’-5’ OAS; 6-16; GBP; 9- 27 and class II HLA-DRa) in response to either IFN-a or IFN-g. Although the expression of these genes varied to some extent in response to the specific inducer, as observed in the parental cell line, the reduction in the mRNA expression levels of these genes in mutant B7 was remarkable (compare Figure 2, lanes 1-3 to 4-6 and 7-9 to 10-12), which agrees with the phenomenon of IFN-partial responsiveness observed by FACS analysis.

Figure 2
- IFN-stimulated gene expression in the parental cell line 2C4 and in the B7 mutant. The expression of 2’-5’ oligo A synthetase (OAS), 9-27 and GBP (lanes 1-6), and class II HLA (DRa), 6-16 and GBP (lanes 7-12) mRNAs in the parental cell line 2C4 and in the B7 mutant was investigated upon IFN-a or IFN-g treatment. Five hundred IU/ml of either IFN-a or IFN-g was added to the cells for 6 h and 18 h, respectively, cytoplasmic RNA was isolated (10 µg per lane), with the specific probe and analyzed by the RNase protection assay as described in Material and Methods. g-Actin was used as an internal standard for loading.

IFN-activated transcriptional complex formation

Upon IFN treatment a number of transcriptional activator complexes are formed depending on the type of ISG promoter (34,38,39). Thus, when the promoter of an ISG possessing a classical ISRE, such as the 9-27 gene, is used as a probe (Figure 3A), two complexes are observed after IFN stimulation in cell extracts prepared from a variety of cell lines (34) including 2C4 cells (this work): i) ISGF3-a, which is activated/formed upon IFN-a treatment (Figure 3A, lane 2); ii) ISGF3-g, represented by p48, which is expressed at a low constitutive level in untreated cells, but whose level increases significantly after either IFN-a or IFN-g treatment (Figure 3A, lanes 1-3). In contrast, the B7 mutant was completely inefficient in terms of ISGF3-a formation upon IFN-a treatment (Figure 3A, lane 5), whereas the expression level of ISGF3-g in untreated cells was similar to that observed in IFN-treated cells (Figure 3A, lanes 4-6).

Figure 3
- IFN-activated transcriptional complex formation. EMSA of ISGF3-a, ISGF3-g, IRF1/2 and p91/p48 in whole cell extracts from IFN-treated 2C4 and B7 cells. Cells were left untreated (U) or treated with 500 IU/ml of either IFN-a or IFN-g for 6 h and 18 h, respectively. Cell extracts were incubated with an end-labelled oligodeoxynucleotide probe containing the promoter region of 9-27 (A, lanes 1-6); 6-16 (B, lanes 7-12) or IRF-1 (C, lanes 13-18) genes. DNA-protein complexes formed are indicated.

By employing the promoter of the 6-16 gene as a probe, another transcriptional complex was found in cell extracts of untreated HT1080 cells, whose expression was increased upon IFN stimulation, mainly IFN-g. Antibody studies identified this complex which comprises IRF1/IRF2 (IFN-regulator factor) proteins (34), and this was also observed for 2C4 cells (HT1080-derived cell line) (Figure 3B, lanes 7-9). On the other hand, the B7 mutant showed a constitutive level of IRF1/IRF2 complex formation regardless of the IFN treatment (Figure 3B, lanes 10-12).

When the promoter of the IRF1 gene was employed as a probe (Figure 3C), a transcriptional complex was detected in cell extracts prepared from 2C4 cells after IFN-g treatment (Figure 3C, lane 15), which was absent upon IFN-a treatment (Figure 3C, lane 14). Antibody characterization identified p91 and p48 (or related proteins) as components of this complex (data not shown), which may act through the GAS element (17,20) or a different motif other than an ISRE (30,40), since the IRF1 promoter is devoid of an ISRE. In contrast, this complex formation was not observed in the B7 mutant. On the other hand, a slightly faster migrating complex was observed in the mutant, which is expressed at a low constitutive level, regardless of the IFN treatment (Figure 3C, lanes 16-18). The significance of this observation remains to be investigated.

All the above DNA-protein complexes formed specifically competed for binding with the homologous oligonucleotide, at 50 molar excess, but did not compete with heterologous oligonucleotides (data not shown).

JAK1 mRNA expression in the B7 mutant

In order to investigate whether the partial responsiveness to both types of IFN in the B7 mutant (FACS analysis and ISGs mRNA expression) is correlated with the level of JAK1 mRNA expression in this cell line, poly A⁺ mRNA was hybridized with the human JAK1 probe. As shown in Figure 4, no detectable JAK1 message was identified in the mutant cell line when compared to the parental cell line 2C4.

Figure 4
- Northern analysis of JAK1 mRNA. Five µg of poly A⁺ RNA from the parental cell line 2C4 and the B7 mutant was hybridized with a human JAK1 cDNA probe as indicated. GAPDH was used as an internal standard for RNA loading.

Discussion

By combining genetic and biochemical approaches, impressive progress has been made towards understanding the nuclear signals triggered by IFN upon ligand-receptor binding (5). A converging approach employing target gene disruption, which affects specific genes such as IFN receptors (41), and regulatory proteins IRF1/IRF2 (42,43), has provided substantial evidence showing how the IFN system operates. On the other hand, structure-function analysis has also provided important information on this emerging picture, for example i) a single amino acid substitution changing tyrosine 701 at the phosphorylation site in the STAT1 to phenylalanine (44) abrogates IFN-a and -g responsiveness, ii) the same substitution at position 690 in the STAT2 interferes with the phosphorylation of STAT1 (31,45), and iii) sequential deletions affecting different domains in the NRPTK-TYK2 restore the loss of kinase function (46).

In the present study I described a recessive mutant cell line, B7, which is partially responsive to both IFN-a and IFN-g. The defect in this mutant was associated with the NRPTK-JAK1, because this cell line is not complemented by fusion with the U4 mutant (JAK1^-), and genetic transfer of human JAK1 cDNA into the cell line enhanced both IFN-a and IFN-g responsiveness. Although the induction of ISGs, upon IFN-a and IFN-g stimulation, was partially observed in this mutant, JAK1 mRNA expression was below detection levels. The nature of mutation(s) affecting JAK1 mRNA expression remains to be further investigated, but one may speculate that it may map either at the promoter region of the gene or at the 3’ end, the latter contributing to the instability of the mRNA. However, the kinetics of ISG stimulation favors the former hypothesis. Whatever the mutation associated with the B7 phenotype, it seems that this mutant is particularly useful to understand cis-acting elements governing JAK1 expression. It is tempting to speculate that even under these circumstances partial ISG stimulation occurs through the JAK/STAT signalling pathway (5).

Transcriptional complex formation, involving IRF-family members (IRF1, IRF2 and p48), which are inducible in the parental cell line 2C4 upon IFN-a and IFN-g treatment, becomes constitutively expressed in the B7 mutant. Whether this phenomenon is directly correlated with the nature of mutation(s) affecting JAK1 expression, or whether they are unrelated events remains to be determined. The possibility that additional mutation(s) contributes to this particular phenotype cannot be ruled out.

Thus, the mutant cell line B7 seems to be of particular importance for the understanding of cis-acting elements governing JAK1 mRNA expression and may represent regulatory mechanisms associated with JAK1 expression and transcriptional activation.

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Acknowledgments

I would like to thank Dr. I.M. Kerr, Imperial Cancer Research Fund, London, for the opportunity to carry out this work in his laboratory. I also thank A. Wilks for providing the human JAK1 cDNA, S. Goodbourn, T. Decker and M. Müller for kindly providing the protection probes, R. Pine, D. Levy and C. Schindler for the antibodies, and Dima Guschin and L.F.L. Reis for a critical reading of the manuscript.

Address for correspondence: C.A. Bonjardim, Laboratório de Vírus, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Bloco F4, 258, 31270-901 Belo Horizonte, MG, Brasil. Fax: [phone]. E-mail: [e-mail]

The author was the recipient of a post-doctoral fellowship from CNPq. Received December 28, 1995. Accepted October 16, 1996.

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  09 Oct 1998
• Date of issue
  Jan 1997

History

• Accepted
  16 Oct 1996
• Received
  25 Dec 1995