Báo cáo khoa học: A novel splicing variant form suppresses the activity of full-length signal transducer and activator of transcription 5A pot

We isolated a novel splicing variant of STAT5A from a cDNA library of the mouse brainstem.. Expression of STAT5A_DE18 was detected in the mouse brainstem, lung and thymus, but not in the

full-length signal transducer and activator of

transcription 5A

Yoshihisa Watanabe1, Masaya Ikegawa2, Yoshihisa Naruse3 and Masaki Tanaka1

1 Department of Cell Biology, Research Institute for Neurological Diseases and Geriatrics, Kyoto Prefectural University of Medicine, Japan

2 Department of Genomic Medical Sciences, Kyoto Prefectural University of Medicine, Japan

3 Department of Anatomy, Medical Education and Research Center, Meiji University of Integrative Medicine, Kyoto, Japan

Introduction

Signal transducers and activators of transcription

(STATs) are cytoplasmic transcriptional factors that

respond to cytokines, growth factors, and peptide

hor-mones [1,2] In mammals, seven members of the STAT

family (STAT1–4, STAT5A, STAT5B, and STAT6)

have been identified STATs are activated through the

phosphorylation of a tyrosine residue located between

a Src homology 2 (SH2) domain and a transactivation

domain Phosphorylated STATs form homodimers, heterodimers, or tetramers, and translocate into the nucleus, where they act as transcription activators [3–6] In addition to the involvement of STATs in immunological intracellular signal transduction, hema-topoiesis, mammary gland development, and lactogen-esis [7], some reports have demonstrated that STAT3 and STAT5 also play important roles in the central

Keywords

brainstem; coaggregation; STAT5A splicing

variant; suppression of STAT5A activity

Correspondence

M Tanaka, Department of Cell Biology,

Research Institute for Neurological Diseases

and Geriatrics, Kyoto Prefectural University

of Medicine, Kawaramachi-Hirokoji,

Kamikyo-ku, Kyoto 602-0841, Japan

Tel ⁄ Fax: +81 75 251 5797

E-mail: mtanaka@koto.kpu-m.ac.jp

Database

The nucleotide sequence for the mouse

STAT5A_DE18 cDNA has been submitted to

the GenBank database under the accession

number EU249543

(Received 4 July 2009, revised 24 August

2009, accepted 1 September 2009)

doi:10.1111/j.1742-4658.2009.07339.x

Signal transducers and activators of transcription (STATs) regulate a vari-ety of cellular functions, including differentiation and proliferation STAT3 and STAT5 are known to play important roles in brain processes, such as energy homeostasis and neuronal development We isolated a novel splicing variant of STAT5A from a cDNA library of the mouse brainstem This variant, STAT5A_DE18, lacked exon 18 and caused a frameshift in the C-terminus, resulting in deletion of a tyrosine phosphorylation site and a transactivation domain Although the frameshift region had no characteris-tic motifs, it was highly serine⁄ threonine-rich and contained a short proline-rich sequence Expression of STAT5A_DE18 was detected in the mouse brainstem, lung and thymus, but not in the mouse cerebrum or cere-bellum We developed a specific antibody against STAT5A_DE18 and investigated the intracellular localization of this variant STAT5A_DE18 showed dot-like structures in the cytoplasm and could not translocate into the nucleus after prolactin treatment STAT5A_DE18 showed a strong ten-dency to aggregate, which led to coaggregation with STAT5A_full-length This coaggregation inhibited the nuclear transport of STAT5A and suppressed prolactin-induced activation of STAT5A

Abbreviations

DAPI, 4¢,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PRL, prolactin; SH2, Src homology 2; STAT, signal transducer and activator of transcription;

STAT5A_FL, signal transducer and activator of transcription 5A_full-length.

nervous system [8–10] Morphological analyses of

STAT5 knockout mice revealed a reduction in the

number of cortical interneurons in the marginal zone,

abnormalities of corticofugal axon projection, and

defective axon guidance [8] Furthermore, neuronal

cell-specific suppressor cytokine signaling-3 (SOCS3)

knockout mice exhibit higher leptin-induced

phosphor-ylation of hypothalamic STAT3, loss of body weight,

and suppression of food intake [9] STAT5 was also

reported to be involved in energy homeostasis

Neu-ron-specific STAT5A and STAT5B knockout mice

develop severe obesity with hyperphagia, impaired

thermal regulation in response to cold,

hyperleptin-emia, and insulin resistance [10] These reports indicate

that STAT3 and STAT5 regulate food intake and

energy utilization in the brain

We previously analyzed the functions and expression

of relaxin 3, which is involved in stress responses and

hyperphagia [11–13] Relaxin 3 is expressed in neurons

of the nucleus incertus of the median dorsal tegmental

pons, and its expression is regulated by

corticotropin-releasing factor [11,14] Relaxin 3-expressing neurons

project into the septum, hippocampus and

feeding-associated regions such as the lateral hypothalamus

area and arcuate nucleus [11] Recently, we determined

the promoter region of the relaxin 3 gene, and found

that it contained a putative binding site for STATs

[14] In an attempt to analyze this transcriptional

regu-lation, we isolated a novel splicing variant of STAT5A,

STAT5A_DE18, from a cDNA library of the mouse

brainstem The variant protein was generated by a

frameshift in the C-terminal region, resulting in

dele-tion of the tyrosine phosphoryladele-tion site and

transacti-vation domain Here, we report that this variant is

predominantly expressed in the brainstem and

coag-gregates with STAT5A_full length (STAT5A_FL)

Furthermore, the expression of this variant suppresses

the activity of STAT5A

Results

Isolation of a novel STAT5A splicing variant

A cDNA encoding STAT5A was cloned from the

mouse brainstem to investigate transcriptional

regula-tion by STAT5 in the brain During the process of

STAT5A cDNA cloning, we found a novel splicing

variant of STAT5A by nucleotide sequencing analysis

The mouse STAT5A gene is composed of 20 exons

that encode a 793 amino acid polypeptide with a

calcu-lated molecular mass of 91 kDa (Fig 1A,

STA-T5A_FL) The novel splicing variant, termed

STAT5A_DE18, lacked the sequence corresponding to

exon 18 of STAT5A The deletion of exon 18 caused a frameshift at Ala688, which led to premature termina-tion at the codon for amino acid 798 (Fig 1A, STA-T5A_DE18) The C-terminal region of the variant lacked the transactivation domain and the tyrosine res-idue (Tyr694) phosphorylated by Janus protein tyro-sine kinase or other tyrotyro-sine kinases, although the DNA-binding domain and SH2 domain remained intact The frameshift region of STAT5A_DE18 (amino acids 688–797) had no characteristic motifs However, it was highly serine⁄ threonine-rich (25.5%) and had a short proline-rich sequence [PQMPE-PAPP(693–701)]

RT-PCR analysis of STAT5A_DE18

To determine the expression of STAT5A_DE18 in mouse tissues, RT-PCR analyses were conducted using specific primers designed within exons 16 and

20 (Fig 1A) The RT-PCR analyses were expected to generate a 387 bp fragment for STAT5A_DE18 and

a 439 bp fragment for STAT5A_FL The PCR prod-uct of STAT5A_FL was detected in multiple tissues, such as the cerebrum, kidney, and liver (Fig 1B)

On the other hand, the PCR product of STA-T5A_DE18 was detected in the brainstem, heart and lung, and thymus (Fig 1B), but not in the cerebrum

or cerebellum Expression of the variant was also observed in the N2a mouse neuroblastoma cell line (Fig 1B)

The genomic structure of STAT5A is highly con-served in humans and mice Human STAT5A has 20 exons, and exon 18 consists of 52 bp, similar to the case for mouse STAT5A Therefore, we examined whether the splicing variant was expressed in the human brainstem To minimize the amplification of STAT5B, the analysis was performed by nested RT-PCR (Fig 2A) Human brainstem cDNAs were synthesized from total RNA extracts of the human pons The first PCR amplification was performed using STAT5A-specific primers designed within the 5¢-UTR and 3¢-UTR Using the first-round PCR products as templates, the second PCR amplification was per-formed The 411 bp product for STAT5A_DE18 was detected in the human pons and mouse brainstem by nested RT-PCR (Fig 2A, lanes 2 and 3) Furthermore,

we reconfirmed that this variant was not expressed in the mouse cerebrum (Fig 2A, lane 1) In the case of human STAT5A_DE18, deletion of exon 18 caused a frameshift at Ala688, which led to a premature stop codon at amino acid 690, resulting in a truncated C-terminus Human STAT5A_DE18 was shorter than the STAT5Ab isoform, encoded by another splicing

variant of STAT5A, and also lacked the

transactiva-tion domain and tyrosine residue (Fig 2B)

Immunoblotting and immunocytochemical

analyses of STAT5A_DE18

To examine the expression and the intracellular

locali-zation of STAT5A_DE18, we produced a polyclonal

antibody against STAT5A_DE18 A rabbit was

immunized with a glutathione S-transferase (GST)–

STAT5A_DE18_C fusion protein, and a polyclonal

antibody against STAT5A_DE18 was affinity-purified

using a thioredoxin–STAT5A_DE18_C-immobilized

column We performed immunoblotting analyses to

examine the specificity of the antibody The antibody

specifically detected exogenous Flag–STAT5A_DE18,

and did not cross-react with STAT5A_FL (Fig 3)

The antibody was subsequently used for

immunoblot-ting and immunocytochemistry of STAT5A_DE18 In

order to examine its intracellular localization, Flag–

STAT5A_DE18 or Flag–STAT5A_FL was transiently

expressed in HeLa cells, and the cells were

immuno-stained with antibodies against STAT5A_DE18 or

STAT5A_FL Flag–STAT5A_DE18 exhibited a dot-like localization in HeLa cells (Fig 4B), whereas Flag– STAT5A_FL was diffusely localized to the cytoplasm and nucleus (Fig 4A) To confirm that this unusual localization of STAT5A_DE18 was not due to over-expression of Flag–STAT5A_DE18 in HeLa cells,

we generated HeLa cells stably expressing a STAT5A_DE18–enhanced green fluorescent protein (EGFP) fusion protein The localization of this fusion protein in the stably transfected cells was similar to that observed in the transiently transfected cells Con-focal laser microscopy revealed that some dot-like structures colocalized with LysoTracker Red, a lyso-somal marker (Fig 4C) However, the dot-like struc-tures did not colocalize with markers for mitochondria

or the endoplasmic reticulum (data not shown) These results indicated that STAT5A_DE18 was localized in the cytoplasm as dot-like structures Next, the nuclear transport of STAT5A_DE18 was investigated using an EGFP fusion protein An expression vector for STAT5A_FL–EGFP or STAT5A_DE18–EGFP was transfected in T47D cells, which endogenously express the prolactin (PRL) receptor [15,16] STAT5A_FL–

1kb ΔΔE18

793 amino acids

STAT5A_FL

Mouse STAT5A gene

Y 694

exon 18 (52 bp)

797 amino acids

STAT5A_ ΔΔE18

688

α-helical

coiled-coil DNA binding SH2 activation

Trans-Pro-rich

(693-701)

G3PDH

FL (439 bp) ΔΔE18 (387 bp)

500 400 300 (bp)

A

B

Fig 1 Gene structure and expression of a novel STAT5A splicing variant (A) The mouse STAT5A gene contains 20 exons The translation initiation codon and stop codon are located in exons 3 and 20, respectively STAT5A_FL encodes a 793 amino acid protein composed of a a-helical coiled-coil domain, a DNA-binding domain, an SH2 domain, and a C-terminal transactivation domain The deletion of exon 18 (52 bp)

in the STAT5A_DE18 mRNA results in a translational frameshift from Ala688 The STAT5A_DE18 protein lacks the Tyr694 phosphorylation site and transactivation domain, and contains a new reading frame with a proline-rich sequence (amino acids 693–701) (B) The expression

of STAT5A_DE18 in mouse tissues was analyzed by RT-PCR PCR was performed using primers designed in exons 16 and 20 (A, arrows) The RT-PCR analyses were expected to generate a 439 bp fragment for STAT5A_FL and a 387 bp fragment for STAT5A_DE18 (upper panel) RT-PCR amplification of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) is indicated as an internal control (lower panel) Lane 1: cere-brum Lane 2: brainstem Lane 3: cerebellum Lane 4: heart Lane 5: lung Lane 6: kidney Lane 7: liver Lane 8: thymus Lane 9: mammary gland Lane 10: adrenal gland Lane 11: N2a cell line Lane 12: STAT5A_FL cDNA Lane 13: STAT5A_DE18 cDNA.

EGFP was translocated into the nucleus after PRL

treatment (Fig 5A,B), whereas the dot-like structures

of STAT5A_DE18–EGFP remained in the cytoplasm

despite PRL treatment (Fig 5C,D)

Aggregate formation by STAT5A_DE18

The characteristic localization of STAT5A_DE18

sug-gested that this protein may form aggregates

There-fore, we investigated whether it could form insoluble

aggregates Flag–STAT5A_DE18 was exogenously

expressed in HeLa or N2a cells, and its solubility in

the detergent Triton X-100 was examined Transfected

cells were separated into 0.5% Triton X-100-soluble

and 0.5% Triton X-100-insoluble fractions, and the

amounts of Flag–STAT5A_DE18 in the fractions were

quantified by immunoblotting with the antibody

against STAT5A_DE18 Flag–STAT5A_DE18 was

recovered only in the insoluble fractions (Fig 6A),

confirming that the dot-like structures were aggregates

of STAT5A_DE18 Furthermore, we examined whether

endogenous STAT5A_FL was also present in these

aggregates, as other STAT isoforms, namely STAT3b

and STAT5b, form heterodimers with their full-length

forms [17,18] To this end, the soluble and insoluble

fractions were analyzed by immunoblotting with the

antibody against STAT5A_FL In the

STAT5A_DE18-expressing cells, endogenous STAT5A_FL was

recov-ered not only in the soluble fractions but also in the insoluble fractions (Fig 6A) These findings were confirmed by immunocytochemistry N2a cells trans-fected with the expression plasmids for STAT5A_ DE18–EGFP and Flag–STAT5A_FL were immuno-stained with the antibody against STAT5A_FL STAT5A_DE18–EGFP formed massive aggregates in N2a cells (Fig 6C, arrows) In EGFP-positive cells, coexpressed STAT5A_FL was also localized to massive

B

A

Fig 2 Human STAT5A_DE18 variant (A)

Expression of STAT5A_DE18 in the human

brainstem was confirmed by nested

RT-PCR In the first PCR amplification, mouse

and human STAT5A were specifically

ampli-fied using primers designed within the

3¢-UTR and 5¢-UTR The second PCR

amplifi-cation was expected to generate a 411 bp

fragment for STAT5A_DE18 and a 463 bp

fragment for STAT5A_FL Lane 1: mouse

cerebrum Lane 2: mouse brainstem.

Lane 3: human pons Lane 4: mouse

STAT5A_FL plasmid Lane 5: mouse

STA-T5A_DE18 plasmid Size markers (M) are

shown on the left (B) Comparisons of the

mouse and human amino acid sequences of

STAT5A_FL, STAT5A_DE18, and STAT5Ab.

The frameshift regions of STAT5A_DE18 are

underlined Shaded letters, bold letters and

the open box show the proline-rich region,

tyrosine phosphorylation residues, and SH2

domains, respectively.

pF la g- C

M V -6 a

pF

g-ST AT 5A

pF

g-ST

5A

18

kDa kDa

pF la g- C

M V -6 a

pF

5A

pF

g-ST

5A

18

Fig 3 Immunoblotting analysis of STAT5A_DE18 The immunor-eactivity of the polyclonal antibody against STAT5A_DE18 was con-firmed by immunoblotting analysis HeLa cells were transfected with a control vector (pFlag–CMV-6a), pFlag–STAT5A_FL or pFlag– STAT5A_DE18 for 48 h Immunoblotting analyses of the cell extracts were performed with polyclonal antibody against STAT5A_FL (left panel) or STAT5A_DE18 (right panel).

aggregates (Fig 6B,D, arrows) This coaggregation was not observed in cells expressing STAT5A_FL alone (Fig 6, arrowheads) These results indicate that expression of STAT5A_DE18 leads to coaggregation with STAT5A_FL

STAT5A_DE18 suppresses STAT5A activity

We investigated whether the aggregation affected cell viability or the transcriptional activity of STAT5A The viabilities of STAT5A_DE18-expressing cells were mea-sured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) and dead cell protease-based cytotoxicity assays In the MTT assays, overexpression

of STAT5A_DE18 did not decrease the viability of N2a cells as compared with the vector control, even at 48 h after transfection (Fig 7A) Furthermore, the dead cell protease-based cytotoxicity assay, which is highly sensi-tive, confirmed that there was no significant difference

in the viability of STAT5A_DE18-expressing cells (Fig 7B) Next, the transcriptional activity of STAT5A was measured in T47D cells, which express STAT5A_DE18 exogenously A luciferase reporter gene joined to the mouse b-casein promoter containing a c-activated sequence was constructed to monitor the activity of STAT5A In vector-transfected T47D cells, PRL stimulation resulted in a 3.3-fold increase in reporter gene expression (Fig 7C) On the other hand, expression of STAT5A_DE18 reduced the activation of PRL-stimulated STAT5A to  2.2-fold (Fig 7C) The expression of STAT5A_DE18 suppressed the PRL-induced activity of STAT5A by 33% Furthermore, we observed nuclear translocation of STAT5A_FL by immunocytochemistry using the antibody against STAT5A_FL T47D cells were transfected with Flag–STAT5A_FL alone (Fig 7D, upper panel) or both Flag–STAT5A_FL and STAT5A_DE18–EGFP (Fig 7D, lower panel), and this was followed by incu-bation with or without PRL for 24 h After fixation, the cells were immunostained with the antibody against

ΔΔE18-EGFP

Flag-FL

ΔΔ

Flag- E18

A

B

C

Fig 4 Immunocytochemistry of STAT5A_DE18 The HeLa cells expressing Flag–STAT5A_FL (A) or Flag–STAT5A_DE18 (B) were analyzed by immunocytochemistry using the antibodies against STAT5A_FL or STAT5A_DE18 (green) Nuclei were stained with DAPI (blue) The small panels on the right represent the immunocy-tochemical images of untransfected HeLa cells Bar: 20 lm (C) HeLa cells were stably transfected with pEGFP–STAT5A_DE18 Liv-ing cells were stained with LysoTracker Red (red), and observed using a confocal laser microscope STAT5A_DE18–EGFP (green) is detected as dot-like structures and localized to the cytoplasm A few dots of STAT5A_DE18–EGFP are colocalized with the lyso-some marker (arrows) Bar: 20 lm.

STAT5A_FL and observed by fluorescence

micros-copy When cells were transfected with

Flag–STA-T5A_FL alone, Flag–STAT5A_FL predominantly

translocated into the nucleus after PRL treatment

(Fig 7D, upper right panel) In cotransfected cells, the

PRL-induced translocation of Flag–STAT5A_FL into

the nucleus was inhibited by its coaggregation with

STAT5A_DE18–EGFP (Fig 7D, lower right panel)

These results are consistent with the hypothesis that

aggregation of STAT5A_DE18 suppresses the

tran-scriptional activity of STAT5A

Discussion

We isolated a novel STAT5A splicing variant from

the mouse brainstem The STAT5A_DE18 variant

lacked the transactivation domain and a tyrosine

residue Many STAT isoforms have previously been

reported to be generated by alternative splicing and

proteolytic processing [17,19] STAT1, STAT3,

STAT4, STAT5A and STAT5B mRNAs are

alterna-tively spliced at the 3¢-end, resulting in the production

of b-isoforms truncated at the transactivation domain

STAT5A b-isoforms and STAT5B b-isoforms are

generated by insertion of intron 18, and lack only

the transactivation domain [18,20] These STAT

b-isoforms are phosphorylated on the tyrosine residue after stimulation by cytokines or hormones, and translocate into the nucleus, but fail to activate transcription [18,21] Unlike the b-isoforms, the STAT5A_DE18 variant was not phosphorylated, because it lacked the tyrosine residue Moreover, STAT5A_DE18 did not translocate into the nucleus

in T47D cells after PRL treatment, indicating a dis-tinct property of STAT5A_DE18 as compared with the b-isoforms However, STAT5A_DE18 clearly suppressed the activity of STAT5A, similar to the case for the b-isoforms It has been reported that the phosphorylated b-isoforms form heterodimers with full-length STATs and decrease their activities [18] These heterodimers can translocate into the nucleus and bind to target sequences on DNA, but fail to activate transcription [18] On the other hand, we demonstrated that the STAT5A_DE18-mediated suppression was caused by coaggregation of STA-T5A_FL and STAT5A_DE18 in cultured cells, although the precise mechanism for the coaggregation remains to be determined It is known that unphos-phorylated STAT5A monomers can dimerize via interactions between their b-barrel (amino acids 332– 470) and four-helix bundle (amino acids 138–331) domains [22] STAT5A_DE18 also contains these domains, suggesting that heterodimers of unphosphor-ylated STAT5A_FL and STAT5A_DE18 are probably formed in the soluble condition prior to their coag-gregation This coaggregation could be the cause of the decrease in functional STAT5A, resulting in suppressed transcription of its downstream genes STAT5Ab is expressed in early myeloid lineages [23], whereas the STAT5A_DE18 variant was expressed

in the mouse brainstem, thymus, and lung Moreover, exon 5 of STAT5A is alternatively spliced by heteroge-neous ribonucleoprotein L-like at the transition from naı¨ve to activated T-cells [24] STAT5A transcripts variously undergo tissue-specific or cell type-specific alternative splicing, suggesting that these variants are involved in specific functions The physiological role of STAT5A_DE18 may involve the regulation of STAT5 function in the brainstem, considering that STA-T5A_DE18 suppresses the activity of STAT5 The functions of STAT5 in the central nervous system have recently been reported Intracerebroventricular admin-istration of granulocyte–macrophage colony-stimulat-ing factor and leptin activated neuronal STAT5 and reduced food intake [10,25] The activation of STAT5 following leptin administration was observed not only

in the hypothalamus but also in areas of the brain-stem, such as the raphe obscurus, raphe pallidus, dorsal motor nucleus of the vagus, and solitary tract

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Fig 5 Nuclear transport analysis of STAT5A_DE18 (A–D)

STA-T5A_FL–EGFP (A, B), STAT5A_DE18–EGFP (C, D), or EGFP (E, F)

were transiently expressed in T47D cells Cells were cultured in

serum-free medium with (+) or without ( )) PRL (10 ngÆmL )1) for

5 h, and observed under a fluorescence microscope Asterisks

indicate nuclei Bar: 20 lm.

nucleus [25] Furthermore, neuron-specific STAT5A

and STAT5B knockout mice develop severe obesity

with hyperphagia, impaired thermal regulation in

response to cold, hyperleptinemia, and insulin

resis-tance [10] These STAT5-mediated functions in the

central nervous system may be controlled by

STA-T5A_DE18 From the results of RT-PCR and

immu-noblotting analyses, however, the expression level of

STAT5A_DE18 seemed to be very low in the normal

brain Furthermore, this variant protein tended to

form aggregates even in stably transfected cells,

suggesting that high-level expression of this variant

might lead to pathological conditions The formations

of aggregates and inclusion bodies in the brain are

pathognomonic features of many neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s dis-ease, and Huntington’s disease [26,27] Recent studies have revealed that mutant huntingtin aggregates inter-act with several transcription finter-actors, such as CREB-binding protein, TATA-CREB-binding protein, and NF-Y, resulting in reduced expression of their target genes [28,29] This inhibition of functional transcription fac-tors may be associated with the normal functions of huntingtin and⁄ or involved in the pathology of Hun-tington’s disease We observed that the transcriptional activation of STAT5A_FL was suppressed by STA-T5A_DE18 aggregates Moreover, it has been reported that defective mutations of STAT5B are involved in

A

Fig 6 Aggregate formation of STAT5A_DE18 and STAT5A_FL (A) pFlag–STAT5A_DE18 or control vector was transfected into N2a (left pan-els) or HeLa (right panpan-els) cells The Triton X-100-soluble (S) and Triton X-100-insoluble (P) fractions were analyzed by immunoblotting with antibody against STAT5A_DE18 (upper panels) or antibody against STAT5A_FL (lower panels) N2a cells were transfected with Flag–STA-T5A_FL and STAT5A_DE18–EGFP (B–D) or Flag–STAFlag–STA-T5A_FL alone (E–G) Flag–STAFlag–STA-T5A_FL was detected with a primary antibody against STAT5A_FL and Alexa Fluor 546-conjugated secondary antibody (B, E) The localization of STAT5A_DE18–EGFP was observed under a fluo-rescence microscope (C, F) The merged image containing DAPI staining (blue) is shown in (D) and (G) Colocalization of the signals appears yellow Bar: 20 lm.

the syndrome of growth hormone insensitivity [30,31].

Considering the specific expression of STAT5A_DE18

in the brainstem and its suppression of STAT5

activ-ity, accumulation of STAT5A_DE18 may be pathogen-ically involved in certain neurological disorders This possibility will be investigated in future studies

Fig 7 Viability and reporter analysis (A, B) N2a or HeLa cells were transfected with pFlag–STAT5A_FL, pFlag–STAT5A_DE18 or control vector (pFlag–CMV-6a) in serum-containing medium After transfection, the cell viability was assessed by MTT (A) and cytotoxicity (B) assays.

As a positive control, the viability of rotenone-treated cells was also measured (C) T47D cells were transfected with pFlag–STAT5A_FL, pFlag–STAT5A_DE18 or control vector (pFlag–CMV-6a), as well as with pCasein-luc and pSV40-Rluc The cells were treated with PRL (10 ngÆmL)1), and this was followed by measurement of the luciferase activities These results are shown as the means ± standard deviation

of three experiments *P < 0.05 versus control cells without PRL stimulation Statistical analyses were performed using ANOVA with Tukey’s HSD post hoc test (D) T47D cells were transfected with pFlag–STAT5A_FL alone (upper panels) or together with pEGFP–STAT5A_DE18 (lower panels) The cells were then incubated with (+) or without ( )) PRL (10 ngÆmL )1) for 24 h, and this was followed by

immunocytochemi-cal analysis using a primary antibody against STAT5A_FL and Alexa Fluor 546-conjugated secondary antibody Nuclei are indicated by asterisks DE18–EGFP–positive and DE18–EGFP-negative cells are indicated by arrows and arrowheads, respectively Bar: 20 lm.

Experimental procedures

Cell culture and transfection

N2a and HeLa cells were routinely maintained in Ham’s

F12 medium containing 10% fetal bovine serum The

human breast cancer T47D cell line was cultured in

RPMI-1640 medium containing 10% fetal bovine serum [32] To

generate a stable cell line, transfected HeLa cells were

passaged into medium containing G418 (400 lgÆmL)1) for

10 days A stable cell line expressing STAT5A_DE18–

EGFP was isolated from the pool of cells by limiting

dilution cloning, and maintained in the same medium

Plasmids

Mouse STAT5A_FL and STAT5A_DE18 cDNAs were

cloned into the pGEM-T easy plasmid (Promega, Madison,

WI, USA) by PCR, using the primers 5¢-CCGTCAGGA

GCCGTCAGAAG-3¢ and 5¢-CCTGGCGCAAGAACTGA

CAC-3¢ For these amplifications, cDNA libraries from the

mouse brainstem and breast were prepared as previously

described [33] Briefly, total RNA was extracted from

C57BL⁄ 6N mouse tissues using TRIzol (Invitrogen,

Carls-bad, CA, USA), and cleaned up with an RNeasy Micro Kit

(Qiagen, Hilden, Germany) cDNAs were synthesized with

a ThermoScript RT-PCR System (Invitrogen), using an

oli-go-dT primer Flag-tagged STA5A_FL and STAT5A_DE18

constructs were generated by PCR using the primers 5¢-GA

ATTCTATGGCGGGCTGGATTCAG-3¢ and 5¢-GTCG

ACCTACAACTGACGTGGGC-3¢ The PCR fragments

were cloned into pGEM-T easy, and this was followed by

subcloning into the EcoRI–SalI site of pFlag–CMV6a

(Sigma-Aldrich, St Louis, MO, USA) STAT5A_DE18–

EGFP was constructed by PCR using the primers 5¢-GA

ATTCGCCACCATGGCGGGCTGGATTC-3¢ and 5¢-CC

CGGGCCAACTGACGTGGGCTCC-3¢, and the resulting

PCR product containing a Kozak sequence was cloned into

pGEM-T easy The EcoRI–SmaI fragment of this plasmid

was subcloned into the EcoRI–SmaI site of pEGFP-N1

(TaKaRa Bio, Otsu, Japan) For construction of a reporter

plasmid (pCasein-luc), the mouse b-casein promoter was

inserted into the firefly luciferase reporter gene by PCR

using the primers 5¢-CTTCATAACTGAGGTTAAAGC

C-3¢ and 5¢-GTCCTATCAGACTCTGTGAC-3¢

PCR analysis

To analyze the expression of mouse STAT5A_DE18, we

designed the specific primers 5¢-CTGCGCTTCAGT

GACTCGGA-3¢ and 5¢-CGTGCCTGGCAACATCCAT

G-3¢, located within exons 16 and 20, respectively

Further-more, we confirmed the expression of mouse and human

STAT5A_DE18 by nested RT-PCR analysis As a first step,

STAT5A containing the 5¢-UTR and 3¢-UTR was specifi-cally amplified from cDNA libraries of the human pons (TaKaRa Bio) and mouse brainstem, using the primers 5¢ -CTGCTCTCCGCTCCTTCCTG-3¢ ⁄ 5¢-CAGAGAGTCTG GAGTCCACG-3¢ and 5¢-CCGTCAGGAGCCGTCAGAA G-3¢ ⁄ 5¢-GACGTGGGCTCCTCACACTG-3¢, respectively The PCR amplification was performed with 35 cycles of

95C for 15 s and 60 C for 180 s Using the resulting PCR products as templates, we examined the existence of exon 18 The primers 5¢-GACCTGCTCATCAACAAGCC -3¢ and 5¢-CATCCATGGTCTCATCCAGG-3¢ were used for a second round of PCR amplification The second round of PCR amplification was performed with 35 cycles

of 95C for 15 s and 60 C for 45 s All PCR amplifica-tions were performed using Z-Taq DNA polymerase (TaKaRa Bio)

Production of an antibody against STAT5A_DE18

To raise mouse STAT5A_DE18-specific antisera, we used the C-terminal region of STAT5A_DE18 (amino acids 688– 797), which was not found in STAT5A_FL A cDNA encoding this unique region, STAT5A_DE18_C, was inserted into the EcoRI–XhoI site of pGEX-6P-1 (GE Healthcare, Little Chalfont, UK) or the EcoRI–SalI site of pThioHisA (Invitrogen), using the primers 5¢-GGAT CCGGTTCGTCAATGCATCC-3¢ and 5¢-CTCGAGCTAC AACTGACGTGGGCTCCTCAC-3¢ Expression of the GST–STAT5A_DE18_C and thioredoxin–STAT5A_DE18_C fusion proteins was induced in Escherichia coli by treatment with 1 mm isopropyl b-d-1-thiogalactopyranoside for 4 h at

37C Inclusion bodies containing these proteins were recovered by centrifugation (20 000 g for 20 min at 4C), and washed with NaCl⁄ Pi containing 0.5% Triton X-100 and 1 mm phenylmethanesulfonyl fluoride The purified inclusion bodies were separated by SDS⁄ PAGE, using a 12.5% gel, and the GST–STAT5A_DE18_C in the poly-acrylamide gel was emulsified with Freund’s Complete Adjuvant (Difco Laboratories, Detroit, MI, USA) To pro-duce an antiserum, a Kbl:NZW rabbit was immunized with this emulsion (Kitayama Labes, Ina, Japan) At 8 weeks after the immunization, the antiserum was recovered and subjected to a titration assay For affinity purification of the antibody against STAT5A_DE18, inclusion bodies of thioredoxin–STAT5A_DE18_C were solubilized in 50 mm phosphate buffer (pH 8.5) containing 8 m urea and 1 mm phenylmethanesulfonyl fluoride, and rapidly refolded by 10-fold dilution in 50 mm phosphate buffer (pH 8.5) The soluble thioredoxin–STAT5A_DE18_C was purified with HiTrap Q HP (GE Healthcare) and immobilized on HiTrap NHS-activated HP (GE Healthcare) The antiserum was loaded onto this affinity column, and this was followed by washing with 1 m NaCl and 1% Triton X-100 The antibody against STAT5A_DE18 was eluted with 100 mm

glycine-HCl (pH 2.8), and this was followed by rapid

neu-tralization The specificity of the purified antibody was

con-firmed by immunoblotting analysis

Immunoblotting

Cells were washed with NaCl⁄ Piand extracted with ice-cold

NaCl⁄ Pi containing 0.5% Triton X-100 and a protease

inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) After

sonication, each sample was fractionated by centrifugation

(20 000 g, 15 min, 4C), and the supernatant was

recov-ered as a soluble fraction The precipitate was washed with

ice-cold NaCl⁄ Pi and recovered as an insoluble fraction

These fractions were extracted in Laemmli buffer and

sepa-rated by SDS⁄ PAGE, using a 12.5% gel; this was followed

by electroblotting onto poly(vinylidene difluoride)

mem-branes (Millipore, Billerica, MA, USA) After blocking, the

membranes were incubated with a polyclonal antibody

against STAT5A_FL (1 : 1000 dilution; Santa Cruz

Bio-technology, Santa Cruz, CA, USA) or the polyclonal

anti-body against STAT5A_DE18 (1 : 500 dilution) for 12 h at

25C The membranes were then incubated with alkaline

phosphatase-conjugated anti-rabbit IgG (1 : 5000 dilution;

Millipore) for 1 h Immunopositive signals were detected

with the nitroblue tetrazolium chloride and

5-bromo-4-chloro-3¢-indolylphosphatase p-toluidine salt reagents

Immunocytochemistry

N2a and HeLa cells (1· 105

) were plated on round cover glasses (13 mm in diameter), and transfected with pFlag–

STAT5A_FL and pSTAT5A_DE18–EGFP, using

Lipofec-tamine LTX (Invitrogen) Cells were cultured for 2 days

after transfection, and fixed with 4% paraformaldehyde in

0.1 m phosphate buffer After being washed with NaCl⁄ Pi,

they were incubated with the antibody against STAT5A_

FL or antibody against STAT5A_DE18 (1 : 100 dilution) in

NaCl⁄ Pi containing 0.1% Triton X-100 overnight at 4C

After three washes with NaCl⁄ Pi, the cells were incubated

with fluorescein isothiocyanate-conjugated or Alexa Fluor

546-conjugated anti-rabbit IgG (1 : 500 dilution;

Invitro-gen) for 4 h at room temperature After washing and

stain-ing with 4¢,6-diamidino-2-phenylindole (DAPI) (Dojindo,

Kumamoto, Japan), the cells on the cover glasses were

mounted on glass slides in an aqueous mounting medium

(GEL⁄ MOUNT; Biomeda, Foster City, CA, USA) and

examined under a fluorescence microscope (Olympus,

Tokyo, Japan) HeLa cells stably expressing

STA-T5A_DE18–EGFP were cultured in glass-bottomed culture

dishes (Iwaki, Tokyo, Japan), and organelles were stained

with MitoTracker Orange CM-H2TMRos, ER-Tracker

Red, and LysoTracker Red DND-99 (Invitrogen) Confocal

laser microscopy was performed on live cells using a

multi-track analysis (LSM 510 META; Carl Zeiss, Oberkochen,

Germany) EGFP was excited using a 488 nm argon laser,

and emission was recorded through a BP 500–530 nm filter Red-emitting dyes were excited with a 543 nm helium–neon laser, and emission was recorded through an LP 560 nm filter

MTT and cytotoxicity assays For MTT and cytotoxicity assays, 1· 104cells were cul-tured in 96-well plates and transfected with control vector, pFlag–STAT5A_FL or pFlag–STAT5A_DE18, using Lipo-fectamine LTX MTT assays were performed using a Cell Counting Kit-8 (Dojindo) according to the manufacturer’s recommendations The absorbances at 429 or 600 nm were measured using a Multiskan Plus (Thermo Fisher Scientific, Waltham, MA, USA) Cytotoxicity assays were performed using a CytoTox-Glo Cytotoxicity Assay Kit (Promega) Luminescence was measured using a GENios (Tecan, Ma¨nnedorf, Switzerland) All experiments were repeated three times

Reporter assay T47D cells were transfected with 0.5 lg of pFlag–STA-T5A_FL, pFlag–STAT5A_DE18, or vector Simultaneously, 0.5 lg of a firefly luciferase reporter plasmid with the mouse b-casein promoter (pCasein-luc) and 0.25 lg of a Renilla luciferase control plasmid (pSV40-Rluc) were cotransfected using the Lipofectamine LTX and PLUS reagents (Invitrogen) At 24 h after transfection, the cells were treated with 10 ngÆmL)1 recombinant human PRL (Cedarlane Laboratories, Burlington, Canada) in serum-free medium for 24 h The cells were lysed with Passive Lysis Buffer (Promega), and the luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a MicroLumat LB96P Luminometer (Berthold Technologies, Bad Wildbad, Germany) All experiments were repeated three times

Acknowledgements

We greatly appreciate the gift of the T47D cell line from Dr J Kitawaki (Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medi-cine) This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No 18500268 to M Tanaka) and a grant from the Research Institute for Neurological Diseases and Geriatrics, Kyoto Prefec-tural University of Medicine (to M Tanaka)

References

1 Darnell JE Jr, Kerr IM & Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to