Bone Marrow Cells Produce a Novel Tshβ Splice Variant That Is

ORIGINAL ARTICLE Bone marrow cells produce a novel TSHb splice variant that is upregulated in the thyroid following systemic virus infection

BH Vincent1, D Montufar-Solis1, B-B Teng2, BA Amendt3, J Schaefer1 and JR Klein1 1Department of Diagnostic Sciences, Dental Branch, The University of Texas Health Science Center, Houston, TX, USA; 2Center for Human Genetics, The Brown Foundation of Molecular Medicine for the Prevention of Human Disease, The University of Texas Health Science Center, Houston, TX, USA and 3Department of Environmental and Genetic Medicine, Texas A&M Health Science Center, Houston, TX, USA

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 50 rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHb splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHb splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHb construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHb construct. The splice variant TSHb protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHb splice variant, but not the native form of TSHb, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHb, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis. Genes and Immunity (2009) 10, 18–26; doi:10.1038/gene.2008.69; published online 28 August 2008

Keywords: isoform; thyrotropin; 5 0 RACE analysis; metabolism; autoimmunity

Introduction of 5 exons, with the coding region located in portions of exons 4 and 5.3 At present, the only evidence for Thyroid-stimulating hormone (TSH), along with lutei- alternative exon splicing for mouse TSHb is in exons 1, 2 nizing hormone, follicle stimulating hormone and and 3,4,5 all of which are outside the TSHb-coding region. chorionic gonadotropin are members of a family of No evidence yet exists for alternative splicing of TSHb glycoprotein hormones. Glycoprotein hormones consist either within the coding region itself, or in ways that of a common a-subunit and unique b-subunits, the latter affect expression of the TSHb protein. being responsible for hormone specificity.1,2 As would The thyroid hormones, thyroxine (T4) and triiodothyr- be expected for molecules that are highly conserved onine (T3), are essential deterministic factors that evolutionarily, there is considerable homology at both influence nearly every aspect of mammalian physiology, the gene and protein levels between human and mouse including basal metabolism, growth, development, mood TSHb. In both species, the TSHb polypeptide consists of and cognition. TSH induces thyroid hormone synthesis 138 amino acids of which 20 amino acids comprise the by promoting the proteolytic conversion of thyroglobulin signal peptide and 118 constitute the mature protein.3 to T4 and T3 in thyroid follicles. Levels of thyroid Overall, there is 82% homology at the nucleic acid level hormones are controlled by the presence of circulating and 88% homology at the amino acid level between pituitary-derived TSH; feedback mechanisms, in parti- human (accession no. NM_000549) and mouse (accession cular T4 levels, modulate TSH synthesis through no. NM_009432) TSHb. The mouse TSHb gene consists hypothalamic-derived thyrotropin-releasing hormone.1,6 The fact that there are extra-pituitary sources of TSH, including TSH produced by cells of the immune system Correspondence: Dr JR Klein, Department of Diagnostic Sciences, has been known for over 20 years.7–9 Although TSH may University of Texas Health Science Center at Houston, Dental have some activity as an immunoregulatory/cytokine- Branch, 6516 MD Anderson Blvd., Houston, TX 77030, USA. 10–13 E-mail: [email protected] like molecule within the immune system, several Received 29 May 2008; revised 4 August 2008; accepted 5 August observations now point to a more direct link between 2008; published online 28 August 2008 immune system TSH and the hypothalamus–pituitary– Novel immune system TSH BH Vincent et al 19 thyroid axis. For example, hypophysectomized mice Results challenged with foreign antigen have increased circulat- 0 ing T4.14 In as much as the elevated levels of T4 in Quantitative real-time PCR (qRT-PCR) and 5 RACE b hypophysectomized mice could not be accounted for analyses identify a novel TSH splice variant expressed through classical pituitary–thyroid circuitry, this sug- in BM hematopoietic cells gested that an extrapituitary source of TSH was The full-length mRNA sequence is shown in Figure 1a, responsible. Additionally, TSH has been shown to be which indicates the positions of the five mouse TSHb actively produced by a subset of CD11b þ bone marrow exons (designated E1–E5), with the translated portion (BM) cells.15 Moreover, following adoptive transfer of beginning with the ATG (bolded) at the second nucleo- CD11 þ BM cells from enhanced green-fluorescent tide of exon 4. The signal peptide is coded for by the protein (EGFP) transgenic mice into non-EGFP mice, region highlighted in blue; the translated portion of the EGFP þ cells traffic to the thyroid where they express mature peptide is highlighted in green; the location of TSH transcripts and secrete TSH.16 the stop codon (TAA) is bolded. As part of our studies aimed at gaining a better Quantitative RT-PCR analysis was performed using understanding of the role of immune system-derived primers targeted to several regions of mouse TSHb TSH, we conducted a comprehensive examination mRNA. A primer set designated ‘470’ was used for PCR of murine BM mRNA using a panel of TSHb primers. amplification with pituitary and BM RNAs. Those These studies revealed significant differences in gene primer sequences, which span the TSHb-coding region, expression in the BM compared with the pituitary in the are targeted to a region in exons 3 (470-50) and 5 (470-30; 50 region of TSHb mRNA in exons 3 and 4. Using 50 Figure 1a). Using the 470 primer set, we consistently rapid amplification of cDNA ends (RACE), we have observed a marked difference (26 987-fold greater) in the identified a novel in-frame TSHb splice variant that is amount of amplified product for pituitary vs BM RNA preferentially expressed in BM cells related to native (Figure 1b). That pattern also held true using five TSHb. This isoform, the first functional alternatively additional upstream primers targeted to regions in spliced form of murine TSHb to be identified, is exon 4 (designated UP1–UP5; Figures 1a and b) with upregulated in thyroid tissues following systemic virus a downstream primer targeted to exon 5 (designated infection. 98-(30)). Conversely, when qRT-PCR analysis was per-

p<0.01

1000000 82952 31871

100000 26987 26201 17805 18301 10000 648 1000 439 100

(pituitary/BM) 10

Relative gene expression 1 98 470 UP1/98 UP2/98 UP3/98 UP4/98 UP5/98 superarray

Figure 1 Characterization of a novel TSHb splice variant produced in BM cells. (a) The full-length TSHb mRNA sequence showing the locations of the five TSHb exons (E1–E5) and the primers used for qRT-PCR. (b) Results of qRT-PCR analyses using pituitary and BM RNA with the primer sets shown in panel a, indicating a statistically significant difference (Po0.01) in gene expression comparing results when upstream primer sequences were targeted to exons 3 and 4 vs upstream primer sequences targeted to exon 5. (c) Sequence of the 50 RACE product generated with the 50 RACE oligo and the downstream TSHb GSP (underlined). The green and red nucleotides are a portion of intron 4 that immediately precedes exon 5 (black). The red portion represents the 27 nucleotides that begin with an ATG codon and continue in- frame through the RACE sequence. (d) Blast results of the 50 RACE sequence reveal complete identity to the mouse TSHb gene in portions of intron 4 (green and red) and exon 5 (black). BM, bone marrow; GSP, gene-specific primer; TSH, thyroid-stimulating hormone; qRT-PCR, quantitative real-time PCR; RACE, rapid amplification of cDNA ends.

Genes and Immunity Novel immune system TSH BH Vincent et al 20 formed using two primer sets targeted to exon 5 (98-50– Relative to the expression of native TSHb, the TSHb splice 98-30, and Superarray; Figure 1a), the fold difference in variant is expressed at low level in pituitary cells but at high gene expression between pituitary vs BM was 648 and level in BM and thyroid cells 439, respectively (Figure 1b). This represented a statisti- Conventional and RT-PCR analysis were performed to cally significant (Po0.01) 62.8-fold reduction (34 019 vs determine whether the novel TSHb splice variant was 543) in the relative gene expression of the ratio of expressed in pituitary, BM, and/or thyroid tissues. For pituitary/BM TSHb expression using upstream primer this, a new primer set designated ‘novel primers’ (Table 1 sequences targeted to exons 3 or 4 compared to primers and Supplementary Figure S1a) was used consisting of a targeted to exon 5 (Figure 1b). 24 nucleotide upstream primer targeted to intron 4, and a We hypothesized that the qRT-PCR differences downstream primer targeted to a sequence located just between BM and pituitary RNAs as a function of the after the TAA stop codon of exon 5. If present, the novel primer target location were because of an alternative TSHb transcript would be amplified using these primers. splicing of the TSHb gene at or near the junction of exons Figure 2a indicates the relative gene expression levels 4 and 5. To obtain a sequence of BM TSHb mRNA using the 470 primer set and the TSHb novel primer set. from that region, the 50 RACE analysis was performed By conventional PCR, transcript levels using the 470 using a highly purified preparation of BM RNA. RACE primers were detectable only in pituitary RNA technology insured that only full-length, nontruncated (Figure 2a, top panel), whereas the novel TSHb transcript mRNAs were used. A sequence, which was consistently was present in all three tissues—the pituitary, the BM, obtained in multiple 50 RACE cDNA clones, is shown in and the thyroid. These findings were confirmed by qRT- Figure 1c. The underlined nucleotide regions are the 50 PCR (Figure 2b), which indicated an overwhelming RACE oligo and the 30 TSHb gene-specific primer preference for the native TSHb using the 470 primer set (Figure 1c). A gene blast search revealed complete with pituitary vs BM or thyroid RNA. In contrast, the homology to a portion of the mouse TSHb gene as amount of levels of qRT-PCR amplification were more shown in Figure 1d. A striking finding from these similar in those three tissues (Figure 2b), implying that experiments was that all of the 50 RACE sequences there is a preferential use of the novel TSHb splice obtained from BM RNA included a portion of intron 4 variant in the BM and thyroid. that was contiguous with exon 5. These sequences are Because the novel TSHb product was generated using highlighted in green and red in Figures 1c and d; the red an upstream primer targeted to a sequence of intron 4, nucleotides designate a part of intron 4 that includes a experiments were carried out to rule out the amplifica- potential ATG (methionine) start codon followed by a tion that had occurred from genomic DNA. Three sequence that codes for nine amino acids (MLRSLFFPQ) experiments confirmed that the amplification was not that are in-frame with TSHb exon 5, beginning at because of genomic DNA. First, if genomic DNA were nucleotide 186 (Figure 1c). Using a program for present, amplification with the 470 primer sets would identifying an open reading frame (ncbi/nlm/nih.gov), yield larger PCR products because of the presence of ATG comprises an open reading frame with a Kozak introns 3 and 4. As seen in Figure 2a (top panel), all three sequence consisting of the ATC nucleotides before the samples were devoid of PCR products larger than the ATG triplet. Thus, these data point to a modified splicing anticipated nucleotide size of 470. Second, when BM mechanism for BM TSHb, and they explain the low levels RNA was tested in a one-step PCR reaction in the in PCR product from BM RNA using upstream primer absence or in the presence of reverse transcriptase using sequences targeted to exons 3 or 4 vs the abundance of the novel upstream primer, a PCR product was obtained product using primers targeted to exon 5. only in the presence of reverse transcriptase and not in

Table 1 Primers used in this study

Primer designation Sequence

470 forward 50-AAGAGCTCGGGTTGTTCAAA-30 470 reverse 50-CACATTTAACCAGATTGCACTG-30 UP1 forward 50-TCCCCGTGCTTTTTGCTCTT-30 UP2 forward 50-GCAAGCAGCATCCTTTTGTA-30 UP3 forward 50-CGTGGATAGGAGAGAGTGTGC-30 UP4 forward 50-TCAACACCACCATCTGTGCT-30 UP5 forward 50-TGCTGGGTATTGTATGACACG-30 98 forward 50-CCGCACCATGTTACTCCTTA-30 98 reverse 50-ACAGCCTCGTGTATGCAGTC-30 50 RACE oligo (forward) 50-CGACTGGAGCACGAGGACACTGAC-30 TSHb GSP (reverse) 50-TGCGGCTTGGTGCAGTAGTTGGTTCTG-30 Novel TSH forward 50-ATCATGTTAAGATCTCTTTTCTTT-30 Novel TSH reverse 50-AACCAGATTGCACTGCTATTGAA-30 Intron primer 4 forward 50-TTGTTCAATGCATTTCTTTTAGC-30 Intron primer 3 forward 50-GAAAGGAAGTGGGGATAAATCA-30 Intron primer 2 forward 50-GATGGGTTAATTGTAGATGTGTGG-30 Intron primer 1 forward 50-CAGAGCTCAGGAGTCCTTTATTG-30 Superarray TSHb (forward and reverse) Category no. PPM30787A Superarray GAPDH (forward and reverse) Category no. PPM02946E

Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GSP, gene-specific primer; TSH, thyroid-stimulating hormone; RACE, rapid amplification of cDNA ends.

Genes and Immunity Novel immune system TSH BH Vincent et al 21 markers pituitary bone marrow thyroid 2000 - pituitary/BM pituitary/thyroid 10,000,000 1000 - 2,752,010 1,000,000 470 500 - primers 100,000 10,377 10,000

1,000 100 - 132 100 2000 - 8 10 1000 -

Relative gene expression 1 500 - Novel TSH 470 primers novel 5' primers primers

100 - - RT blank + RT + RT

200 - GAPDH 100 - Figure 2 Native TSHb is expressed at high levels in the pituitary but not in the bone marrow or in the thyroid, whereas the novel TSHb splice variant is expressed in all three tissues. (a) PCR and agarose gel electrophoresis analysis of pituitary, bone marrow and thyroid RNA using the 470 primer set that spans the TSHb-coding region yielded a product that was evident only from pituitary RNA. In contrast, using primers (see Supplementary Figure S1a) for the novel TSHb splice variant, a PCR product of the anticipated size was obtained using RNA from all three tissues. (b) qRT-PCR was used to compare the ratio of the 470 PCR product and the novel splice variant product of pituitary/ BM and pituitary/thyroid. Using the 470 primer set, there was an extremely high preference for expression of native TSHb in the pituitary relative to the BM and thyroid. Using the novel primer set, however, the ratio of pituitary/BM and pituitary/thyroid was substantially lower. (c) To confirm that amplification using the novel primer set had not occurred from the genomic DNA, a one-step PCR reaction was performed in which reverse transcriptase was included or omitted. Note that amplification occurred only in the presence of reverse transcriptase, thus excluding the possibility that contaminating genomic DNA was present. (d) The presence of contaminating genomic DNA was also ruled out using four upstream primers targeted to intron 4 (see Supplementary Figure S1b). Amplification using those primers with the 98-30 primer with genomic DNA yielded four PCR products of the anticipated sizes. In contrast, PCR products were obtained from BM RNA only with intron primers 1 and 2, both of which target a region near the 50 RACE start site. These findings also confirm that the data shown in Figures 1c and d accurately reflect the 50 RACE start site. BM, bone marrow; TSH, thyroid-stimulating hormone; qRT-PCR, quantitative real-time PCR; RACE, rapid amplification of cDNA ends.

3.5 3 2.5 2 1.5 1 0.5 0 0 10 20 30 40 50 60 70 80 90 100

50 40 30

20 kDa non-transfected - 19 TSHb (ug/ml) 10 - 15 0 - 6 native splice Lac-Z variant Figure 3 Amino-acid composition of the novel TSHb splice variant. (a) Predicted amino-acid sequence of the novel TSHb slice variant, consisting of a 9-amino acid signal peptide (green residues) and an 84- amino-acid polypeptide (red residues). (b) Location of the novel TSHb isoform (red residues) within the 118-amino-acid sequence of the mature native TSHb molecule. (c) Secondary structure analysis of the novel TSHb polypeptide. The gray line is the hydrophobic momentum index; the red line is the transmembrane helix momentum; the blue line is the beta preference index. Note the high hydrophobic momentum index and the high transmembrane helix momentum of the first 7–9 amino acids that would comprise the signal peptide. (d)TSHb is secreted into the media from CHO cells transfected with native or splice variant TSHb constructs, indicating that both forms of TSHb are produced as secreted proteins. Control CHO cells transfected with LacZ had no detectable TSHb. Data are mean values±s.e.m. of three replicate samples. (e) Cell lysates from nontransfected CHO cells were nonreactive by immunoprecipitation. Immunoprecipitation of cell lysates from CHO cells transfected with the native TSHb construct produced a 17 kDa product; a 8 kDa product was precipitated from lysates of CHO cells transfected with the novel TSHb construct. TSH, thyroid-stimulating hormone. the absence of reverse transcriptase (Figure 2c). Third, yielded products of the correct size from genomic using four upstream primer sequences targeted to DNA, but only the two primers for regions at the regions of intron 4 (Supplementary Figure S1b), all beginning of the RACE sequence yielded products from

Genes and Immunity Novel immune system TSH BH Vincent et al 22 BM RNA (Figure 2d). The latter not only confirmed the in vitro.18 The cAMP response induced by recombinant lack of genomic DNA in RNA preparations, but it also TSHb proteins, although low, were generally in line with validated the 50 RACE findings as accurately defining the some reports of cAMP responses from FRTL-5 cells.19 A beginning of the BM TSHb splice variant. lower cAMP response from FRTL-5 cells also may be because of poor binding of mouse TSH to rat FRTL-5 The novel TSHb protein is an actively secreted protein cells, an interpretation that is supported by the fact that The physiochemical characteristics of the novel TSHb bovine TSH generated a stronger cAMP response from polypeptide predicted from the nucleotide sequence is FRTL-5 cells (8.83 and 7.75 pmol mlÀ1 cAMP following shown in Figure 3. This consists of a 9-amino-acid leader stimulation with 10À7 and 10À9 M bovine TSH, respec- sequence followed by an 84-amino acid polypeptide of tively). Collectively, these findings confirm that the the mature protein molecule coded for by exon 5 up to polypeptide made from the TSHb splice variant is the TSHb stop codon (Figure 3a). The difference between biologically active with regard to its ability to induce a the novel TSHb polypeptide (Figure 3b, red) and the cAMP signal. native TSHb molecule is the lack of amino acids coded Given our previous studies demonstrating that TSH- for by exon 4. The secondary structure of the novel TSHb producing BM cells migrate to the thyroid,16 we were splice variant is shown in Figure 3c, Note the high interested in determining whether immune challenge by hydrophobic moment index (gray line; 43.0) and the virus would result in changes in intrathyroidal levels of high transmembrane helix preference (red line; 2.0) for native and/or splice variant TSHb. C57BL/6 mice were the first 9-amino acids, thus favoring a transmembrane infected i.p. with 107.5 plaque forming units (PFU) T3D location and a likely signal peptide function. Impor- reovirus. The rationale for using reovirus was on the tantly, cell-free supernatants from Chinese hamster ovary basis of studies demonstrating altered thyroid function (CHO) cells transfected with native and splice variant following reovirus infection.20,21 Thyroid tissues were TSHb constructs had high levels of TSHb as detected by isolated 48 h later; RNA was extracted, and qRT-PCR was reactivity with an antimouse TSHb-specific monoclonal performed using the 470 and the novel TSHb primer sets. antibody17 (Figure 3d). Supernatants from control CHO Systemic virus infection did not alter the level of native cells transfected with a LacZ construct were nonreactive TSHb gene expression in the thyroid related to that of with the anti-TSHb antibody. As CHO cells were not noninfected mice; however, there was a statistically transfected with the TSHa gene, it is assumed that the significant increase in gene expression of the TSHb splice observed activity is due to TSHb alone. These findings variant in the thyroid of virus-infected mice (Figure 4b). collectively indicate that the novel TSHb product is These findings suggest that the intrathyroidal host actively secreted from cells. response is linked to the TSHb splice variant, but not Immunoprecipitation was performed using an anti- the native form of TSHb. TSHb antibody directed to a portion of the molecule that is shared by both native and novel TSHb to confirm that transfected CHO cells produced TSHb of the correct Discussion molecular size. Cell lysates from nontransfected CHO cells were nonreactive by immunoprecipitation This study has identified the first functionally active (Figure 3e). Immunoprecipitation of cell lysates from splice variant of mouse TSHb. The novel nature of this CHO cells transfected with the native TSHb construct isoform, involving the retention of a portion of intron 4, produced a 17 kDa product; a 8 kDa product was makes it particularly intriguing. Splice variants that precipitated from lysates of CHO cells transfected with incorporate intronal pieces,22–27 though once thought to the novel TSHb construct (Figure 3e). be uncommon, appear to occur at a higher frequency than previously believed, possibly occurring in upwards The TSHb splice variant is biologically active and is of 15% of human and mouse genes.28,29 However, most upregulated in the thyroid during systemic virus infection such splicing events are associated with disease condi- Recombinant native and splice variant proteins were also tions or with tumor cells, and they frequently result in used to evaluate their ability to induce a cAMP response truncated proteins or aborted translation because of the from the mouse alveolar macrophage (AM) cells and rat generation of a stop codon.30 The splice variant described FRTL-5 cells. As described in the Materials and methods, here consists of a portion of intron 4 and it includes all of cAMP activity was assayed in cell supernatants after 3 h the coding region of exon 5, but none of exon 4, thereby of TSH stimulation. Mouse AM cells were confirmed to coding for a polypeptide that corresponds to 71.2% of the express the TSH receptor by RT-PCR (data not shown). mature native TSHb molecule. Interestingly, exon 5 of Culture of AM cells with recombinant native and splice TSHb, the coding portion retained in the novel TSHb variant TSHb induced a cAMP responses with native slice variant, is important for the biological function of TSHb having peak activity at a concentration of 10À10 M, TSH as it includes an 18-amino-acid ‘seatbelt’ region and splice variant TSHb having peak activity at a (CNTDNSDCIHEAVRTNYC) that is used for attachment concentration of 10À6 M (Figure 4a). Differences in those to the a-subunit. This suggests that the splice variant responses as a function of TSH concentration may reflect may retain the ability to function as a heterodimeric differences in receptor-binding activities. Future studies complex. are planned to examine this possibility. Using an adoptive cell transfer system involving EGFP Using FRTL-5 cells, both native and the novel TSHb BM cells injected into non-EGFP mice, we have demon- splice variant induced dose-dependent cAMP responses strated that some BM cells consistently traffic to the (Figure 4b). Moreover, the optimal molar concentrations thyroid of healthy animals.16 Trafficking also occurred for these responses (10À10–10À12) were typical of TSH using CD11b þ EGFP cells that were matured from BM concentration used by others to induce cAMP responses cells by in vitro culture for 6 days with granulocyte-

Genes and Immunity Novel immune system TSH BH Vincent et al 23 18 Native TSH Media 16 Splice variant TSH Forskolin 14 12 * 10 * 8 6

cAMP (pmol/ml) 4 2 0 -4 -6 -8 -10 -12 -4 -6 -8 -10 -12 0 -6

Log10M

470 primers novel primers Native TSH Media 8 3.5 p< 0.01 Splice variant TSH Forskolin 7 3 p=0.007 6 2.5 5 2 * * 4 1.5 3 1 cAMP (pmol/ml) 2 N.S. 0.5 1 0 gene expression Relative 0 -8 -10 -12 -8 -10 -12 0 -5 non- reo3 non- reo3 log10M infected infected Figure 4 Recombinant novel TSHb splice variant is capable of delivering a cAMP signal and is upregulated in the thyroid following systemic virus infection. (a) cAMP response of AM cells cultured with log10 dilutions of recombinant native TSHb, splice variant TSHb, media, or forskolin at the concentration indicated. Both forms of TSHb elicited a cAMP response in a dose-dependent fashion. (*Po0.05 compared with other molar concentrations for that form of TSH). Data are mean values±s.e.m. of four replicate samples. (b) FRTL-5 cells, seeded into 24-well plates as described in the Material and methods, were cultured with log10 dilutions of recombinant native TSHb, splice variant TSHb, media or forskolin at the concentrations indicated. Both forms of TSHb elicited a cAMP response in a dose-dependent fashion. (*Po0.01 compared with other molar concentrations for that form of TSH). Data are mean values±s.e.m. of three replicate samples. (c) qRT-PCR analysis of RNA from thyroid tissues 48 h postreovirus infection using the 470 and novel primer sets. Note the statistically significant increase in the novel TSHb splice variant gene expression in the thyroid of infected mice compared with noninfected mice and the lack of change in the thyroid in gene expression of native TSHb in the thyroid during virus infection as identified with the 470 primers. Data are mean values±s.e.m. of three replicate values. In each case, gene expression of virus-infected mice was compared with that of noninfected mice, the latter being designated as a gene expression level of 1.0. TSH, thyroid-stimulating hormone; qRT-PCR, quantitative real-time PCR. macrophage colony-stimulating factor and injected into activity. On the one hand, it could function agonistically nonirradiated, non-EGFP recipient mice; twenty-five to elicit a thyroid hormone response leading to the days later, there was an abundance of EGFP þ cells increases in T3 and T4 synthesis and an upregulation in surrounding thyroid follicular areas.16 In those studies, cellular and physiologic metabolic activity. The fact that donor EGFP þ cells were documented to produce TSHb the TSHb splice variant was capable of inducing a cAMP in situ by immunocytochemical staining. At that time, we response provides some support for this possibility. were unaware of the splice variant form of TSHb, and Conversely, it is possible that the TSHb splice variant has the anti-TSHb antibody used was generated to 12 amino- an antagonistic activity, which serves to restrict thyroid acid residues of TSHb that are coded for by exon 5. hormone synthesis by binding to and competing for TSH As such, that antibody would not discriminate receptor signaling. Clearly, considerably more work will between native and splice variant forms of TSHb.In be needed to discriminate between these two possibilities this study, it is clear that the novel TSHb splice variant is in the context of both normal physiological conditions preferentially expressed in the BM and the thyroid and in various pathological states. However, because the (Figures 2b and c), and that gene expression increases immune system is vested with the capacity to detect a in the thyroid following systemic virus infection wide range of threats to the health of the organism, (Figure 4b). As the BM cells appear to be a primary including challenge because of acute and chronic source of TSHb in the thyroid,15,16 the most likely infection, malignant conditions and debilitating condi- interpretation of our findings is that the higher level of tions, such as coronary disease, the network described TSHb gene expression is either because of an increase in here provides an additional source of information that is TSH synthesis by resident BM cells in the thyroid, or not typically incorporated in sensory processing by the because of the increased trafficking of BM cells to the neuroendocrine system. Microregulation of thyroid thyroid during infection. hormone activity by immune system TSH may be There are several potential ways in which immune important for adjusting to altered metabolic demands system TSH might serve to regulate thyroid hormone during times when energy conservation is needed.

Genes and Immunity Novel immune system TSH BH Vincent et al 24 Finally, it should be noted that a system such as this competent Escherichia coli were transformed with 4 mlof could have negative consequences for the host, particu- the 50 RACE PCR product; transformed cells were larly if the TSHb splice variant displays an enhanced selected on the basis of kanamycin resistance. The clones potential for immunogenicity relative to native TSHb. were grown overnight in Luria broth in the presence of Local intrathyroidal production of the TSHb splice kanamycin. Plasmid DNA was purified using a Qiagen variant under the scenario described above may inad- QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, CA, vertently lead to the generation of anti-TSH autoantibo- USA). Sequencing was performed by Seqwright Inc. dies and autoimmune thyroiditis, as it can occur (Houston, TX, USA) using M13 primers. following systemic virus infection.21 To obtain recombinant proteins, native TSHb and splice variant TSHb DNAs were subcloned into pcDNA3.1/V5-His-TOPO vectors. Plasmid DNA was Materials and methods obtained using standard methods. CHO cells grown in serum-free CHO-CD media (Sigma-Aldrich, St Louis, Mice MO, USA) were transfected with native or novel plasmid The 6–8-week-old female C57BL/6 mice were purchased DNA using an Amaxa electroporator (Amaxa Biosys- from Harlan Sprague–Dawley (Indianapolis, IN, USA). tems, Gaithersburg, MD, USA). Cells were selected for Mice were used in accord with the University of Texas stable tranfectants by continuous culture in 1.2 mg mlÀ1 Health Science Center institutional animal welfare neomycin. A total of 107 cells were used to purify guidelines. His-tagged recombinant proteins using a NI-NTA Fast Start Kit (Qiagen). Estimate of protein concentration was RNA isolation and PCR analyses determined using a Coomassie Plus-200 Protein assay Quantitative RT-PCR analysis was performed using RNA (Pierce, Rockford, IL, USA). Recombinant proteins were isolated with an RNAeasy Protect Mini Kit-50 (Qiagen, stored at À80 1C in the presence of 0.5% bovine serum Valencia, CA, USA); samples were treated with DNase albumin for stabilization. using an RNase-Free DNase Set-50 (Qiagen) according to the manufacturer’s protocols. RNA concentrations were cAMP assay determined at A260. Primer sets were purchased from FRTL-5 cells were obtained from American Type Culture IDT Technologies (Coralville, IA, USA) or Superarray Collection (Manassas, VA, USA). The mouse AM cells Bioscience Corp. (Frederick, MD, USA). qRT-PCR was line, AMJ2-C8 (American Type Culture Collection) was a performed on 100 ng total RNA using an iScript One-step gift from Dr Chinnaswamy Jagannath, Department of RT-PCR kit with SYBR Green (Bio-Rad, Hercules, CA, Pathology, The University of Texas Health Science Center USA). A blank sample with RNase-free water was used at Houston. FRTL-5 cells were grown in Dulbecco’s for primer controls. Amplification was performed in 96- modified eagle media supplemented with 10% FBS, well thin wall plates sealed with optical quality film in a 2mML-glutamine, 10 mgmlÀ1 insulin, 10 nM hydrocorti- Mini-Opticon (Bio-Rad) with a program of 10 min at sone, 5 mgmlÀ1 transferin, 10 ng mlÀ1 gly-his-lys-acetate, 50 1C for cDNA synthesis, 5 min at 95 1C for reverse 10 ng mlÀ1 somatostatin and 10 mU mlÀ1 bovine TSH transcriptase inactivation, followed by 45 cycles of 95 1C (Sigma-Aldrich). AM cells were grown in defined for 10 s and 55 1C for 30 s for data collection. A melt curve serum-free hybridoma medium (Gibco-Invitrogen, 1 was performed using a protocol of 1 min at 95 C and Carlsbad, CA, USA) supplemented with 2 mML-gluta- 1 min at 55 1C, increasing the temperature in 0.5 1C mine. Five to seven days before stimulation with native increments for 80 cycles of 10 s each. Real-time PCR data and splice variant TSHb, media lacking bovine TSH were quantified using the 2ÀDDCt method of Livak and was used for FRTL-5. FRTL-5 cells were grown to 80% Schmittgen;31 samples were normalized to respective confluency in 24-well Corning tissue culture plates GAPDH values using a Gene Expression Macro Version (Fisher Scientific, Pittsburgh, PA, USA); AM cells were 1.1 program (Bio-Rad). grown to a density of 1–2 Â 106 mlÀ1. As described by others, 18 FRTL-5 cells were washed twice with Hank’s 0 The 5 RACE, cloning, sequencing and generation of balanced salt solution containing 0.4% BSA, 220 mM recombinant proteins sucrose and 1 mM isobutylmethylxanthine (Sigma). AM The 50 RACE was performed using a GeneRacer Kit cells were washed and seeded at a density of 1 Â106 cell

(Invitrogen, Carlsbad, CA, USA). Briefly, highly pure per ml in 1.5 ml eppendorf tubes. Log10 M dilutions of RNA isolated from BM cells was dephosphorylated with recombinant native TSHb, splice variant TSHb, forskolin calf intestinal phosphatase to insure that only full-length or media for control cultures was added for 3 h at 37 1Cin

nontruncated mRNA was used. RNA was treated with a humidified 5% CO2 incubator. Cell-free supernatants tobacco acid pyrophosphatase to remove the 50 cap were collected and assayed for cAMP activity using structure from intact full-length mRNA. A 50 RACE a commercial assay kit (R&D Systems, Minneapolis, Oligo provided with the kit was ligated to the 50 end of MN, USA). the mRNA. The ligated mRNA was reverse transcribed using SuperScript III reverse transcriptase to create a Immunoprecipitation RACE-ready first strand cDNA. The cDNA was ampli- A total of 2 Â 106 nontransfected CHO cells, CHO cells fied using Platinum Pfx DNA polymerase with the 50 transfected with native TSHb and CHO cells transfected RACE Oligo primer and a TSH gene-specific primer. The with splice variant TSHb were lysed on ice for 15 min in a RACE PCR products were purified using a simple buffer consisting of 50 mM Tris-HCL (pH 7.4), 150 mM nucleic acid pres (SNAP) column provided with the kit. NaCl, 1 mM EGTA, 2 mgmlÀ1 aprotinin 1 mgmlÀ1 leupep- PCR products were cloned into a pCR4BLUNT-TOPO tin and 1 mM phenylmethyl sulfonyl fluoride (Sigma, all vector using a TOPO cloning kit (Invitrogen). Chemically reagents). Lysates were clarified by high-speed centrifu-

Genes and Immunity Novel immune system TSH BH Vincent et al 25 gation in a microfuge for 5 min. Supernatants were 6 Shupnik MA. Thyroid hormone suppression of pituitary collected and precleared by end-over-end mixing over- hormone gene expression. Rev Endocr Metab Disord 2000; 1: night with 50 ml of protein G-plus agarose (Santa Cruz 35–42. Biotechnologies, Santa Cruz, CA, USA). Protein G was 7 Kruger TE, Smith LR, Harbour DV, Blalock JE. Thyrotropin: an removed from lysates by three successive centrifugation. endogenous regulator of the in vitro immune response. Lysates were reacted by end-over-end mixing for 1 h at J Immunol 1989; 142: 744–747. 4 1C with 25 ml of anti-TSHb antibody (N-19) (Santa Cruz) 8 Smith EM, Phan M, Kruger TE, Coppenhaver DH, Blalock JE. adsorbed to protein G. Protein G immune complexes Human lymphocyte production of immunoreactive thyrotropin. Proc Natl Acad Sci USA 1983; 80: 6010–6013. were washed with lysis buffer and suspended in 30 mlof 9 Harbour DV, Kruger TE, Coppenhaver D, Smith EM, Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) Meyer III WJ. Differential expression and regulation of containing 5% b-mercaptoethanol, boiled for 5 min and thyrotropin (TSH) in T cell lines. Mol Cell Endocrinol 1989; 64: electrophoresed though a precast 12% polyacrylamide 229–241. gel (Bio-Rad). Gels were fixed and exposed to silver 10 Kelley KW, Weigent DA, Kooijman R. Protein hormones and staining using the reagents and methods of M Barton immunity. Brain Behav Immun 2007; 21: 384–392. Frank (http://omrf.ouhsc.edu/~frank/SILVER.html). 11 Fabris N, Mocchegiani E, Provinciali M. Pituitary-thyroid axis and immune system: a reciprocal neuroendocrine-immune interaction. Horm Res 1995; 43: 29–38. 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