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

Abstract

Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta 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 TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.

Figure 1

Characterization of a novel TSHβ splice variant produced in BM cells. (a) The full-length TSHβ mRNA sequence showing the locations of the five TSHβ 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 (p<0.01) in gene expression comparing results when upstream primers sequences were targeted to exons 3 and 4 vs. upstream primer sequences targeted to exon 5. (c) Sequence of the 5′ RACE product generated with the 5′ RACE oligo and the downstream TSHβ 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 5′ RACE sequence reveal complete identity to the mouse TSHβ gene in portions of intron 4 (green and red) and exon 5 (black).

Figure 2

Native TSHβ is expressed at high levels in the pituitary but not in the bone marrow or in the thyroid, whereas the novel TSHβ splice variant is expressed in all three tissues. (α) PCR and agarose gel electrophoresis analysis of pituitary, bone marrow, and thyroid RNA using the 470 primer set that spans the TSHβ coding region yielded a product that was evident only from pituitary RNA. In contrast, using primers (see S1a) for the novel TSHβ 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 TSHβ 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 genomic DNA, a one-step PCR reaction was done 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 S1b). Amplification using those primers with the 98-3′ 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 5′ RACE start site. These findings also confirm that the data shown in Figure 1c and d accurately reflect the 5′ RACE start site.

Figure 3

Amino acid composition of the novel TSHβ splice variant. (a) Predicted amino acid sequence of the novel TSHβ slice variant, consisting of a nine amino acid signal peptide (green residues) and an eighty-four amino acid polypeptide (red residues). (b) Location of the novel TSHβ isoform (red residues) within the 118 amino acid sequence of the mature native TSHβ molecule. (c) Secondary structure analysis of the novel TSHβ polypeptide. The grey 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) TSHβ is secreted into the media from CHO cells transfected with native or splice variant TSHβ constructs, indicating that both forms of TSHβ are produced as secreted proteins. Control CHO cells transfected with LacZ had no detectable TSHβ. Data are mean values ± SEM of three replicate samples. (e) Cell lysates from non-transfected CHO cells were non-reactive by immunoprecipitation. Immunoprecipitation of cell lysates from CHO cells transfected with the native TSHβ construct produced a 17 kDa product; a 8 kDa product was precipitated from lysates of CHO cells transfected with the novel TSHβ construct.

Figure 4

Recombinant novel TSHβ 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 TSHβ, splice variant TSHβ, media, or forskolin at the concentration indicated. Both forms of TSHb elicited a cAMP response in a dose-dependent fashion. (*p<0.05 compared to other molar concentrations for that form of TSH). Data are mean values ± SEM 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 TSHβ, splice variant TSHβ, media, or forskolin at the concentrations indicated. Both forms of TSHβ elicited a cAMP response in a dose-dependent fashion. (*p<0.01 compared to other molar concentrations for that form of TSH). Data are mean values ± SEM of three replicate samples. (c) qRT-PCR analysis of RNA from thyroid tissues 48 hrs post-reovirus infection using the 470 and novel primer sets. Note the statistically-significant increase in the novel TSHβ splice variant gene expression in the thyroid of infected mice compared to non-infected mice, and the lack of change in the thyroid in gene expression of native TSHβ in the thyroid during virus infection as identified with the 470 primers. Data are mean values ± SEM of three replicate values. In each case, gene expression of virus infected mice was compared to that of non-infected mice, the latter being designated as a gene expression level of 1.0.