Bone Marrow Cells Produce a Novel Tshβ Splice Variant That Is
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Genes and Immunity (2009) 10, 18–26 & 2009 Macmillan Publishers Limited All rights reserved 1466-4879/09 $32.00 www.nature.com/gene 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.