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The transcription factor GATA2 regulates gene expression in several cells and tissues, including hematopoietic tissues and the central nervous system. Recent studies revealed that loss-of-function mutations in GATA2 are associated with hematological disorders. Our earlier in vitro studies showed that GATA2 plays an essential role in the hypothalamus–pituitary–thyroid axis (HPT axis) by regulating the genes encoding prepro-thyrotropin-releasing hormone (preproTRH) and thyroid-stimulating hormone β (TSHβ). However, the effect of GATA2 mutants on the transcriptional activity of their promoters remains unelucidated. In this study, we created five human GATA2 mutations (R308P, T354M, R396Q, R398W, and S447R) that were reported to be associated with hematological disorders and analyzed their functional properties, including transactivation potential and DNA-binding capacity toward the preproTRH and the TSHβ promoters. Three mutations (T354M, R396Q, and R398W) within the C-terminal zinc-finger domain reduced the basal GATA2 transcriptional activity on both the preproTRH and the TSHβ promoters with a significant loss of DNA binding affinity. Interestingly, only the R398W mutation reduced the GATA2 protein expression. Subsequent analysis demonstrated that the R398W mutation possibly facilitated the GATA2 degradation process. R308P and S447R mutants exhibited decreased transcriptional activity under protein kinase C compared to the wild-type protein. In conclusion, we demonstrated that naturally occurring GATA2 mutations impair the HPT axis through differential functional mechanisms in vitro.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the β chain (TSHβ). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHβ and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRβ2, into kidney-derived CV1 cells. We found that liganded TRβ2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRβ2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LβT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRβ2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRβ2. The inhibition by T3-bound TRβ2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHβ gene.

Mutations in the POU1F1 gene cause defects in the expression of the genes encoding thyroid-stimulating hormone (TSH)-β subunit, growth hormone (GH), and prolactin (PRL). Here, we characterized 15 missense and nonsense mutations. Protein stability was reduced in the P14L, P24L, F135C, K145X, F233S and E250X mutants. Transactivation by 15 mutants in the TSHβ promoter was moderately correlated with that of the PRL promoter. Based on their transcriptional activity, we classified them into three groups: group I, equivalent to the wild type; group II, partial; and group III, substantially lost. A review of case reports on four patients with group II mutations revealed that TSH deficiency manifested after recombinant GH therapy. A transcription factor, GATA2, is the main activator in the TSHβ gene, while POU1F1 protects its function from inhibition by the suppressor region (SR). We found that the SR is critical for the pathogenesis of TSH deficiency. The transactivation of the TSHβ promoter by the K216E mutant was equivalent to that of wild-type POU1F1; however, that of the PRL promoter was low, while the opposite was found in the R271W mutant. The functional property of K216E suggests that the interaction of POU1F1 with GATA2 may not always be necessary for the activation of the TSHβ promoter.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) [beta]2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin [beta] subunit (TSH[beta]) gene suggest that there is a tethering mechanism, whereby liganded TR[beta]2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSH[beta] gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TR[beta]2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TR[beta]2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TR[beta]2 interferes with this activation via a tethering mechanism as in the case of the TSH[beta] gene.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the [beta] subunit of TSH (TSH[beta]). We previously reported that T3-dependent repression of the TSH[beta] gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived T[alpha]T1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with T[alpha]T1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TR[beta]2. The inhibition by T3-bound TR[beta]2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TR[alpha]1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSH[beta] gene.

T3 inhibits thyrotropin-releasing hormone (TRH) synthesis in hypothalamic paraventricular nucleus (PVN). Although T3 receptor (TR) β2 is known to mediate the negative regulation of prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene indicate the tethering mechanism, where T3-bound TRβ2 interferes with the function of the transcription factor GATA2, which is essential for TSHβ expression. Interestingly, the transcription factor Sim1, a determinant of PVN differentiation in hypothalamus, is reported to induce the expressions of TRβ2 and GATA2. Indeed, our immunohistochemistry revealed the expression of GATA2 in the TRH neuron of the rat PVN. According to the experimental report with transgenic mice, the DNA sequence from nt. -547 to nt. +84 is sufficient for the expression of the prepro-TRH gene in PVN. Using the CAT reporter gene harboring this region, we found that this promoter is activated by GATA2 approximately 6-fold in CV1 cells. The deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. T3-dependent repression was maintained after the mutation of the putative negative T3 responsive element (site4). Although the melanocortin 4 receptor signaling is known to stimulate the prepro-TRH promoter via protein kinase A pathway in the PVN, inhibition by T3 was dominant over the 8-bromo-cAMP-induced activation. We observed the in vivo recognition of GATA-RE by GATA2 using chromatin immunoprecipitation assay with CA77 cells, which express endogenous TRH. The electrophoretic mobility shift assay also demonstrated that GATA2 bound to oligonucleotide containing the GATA-RE. These results suggest that, as in the case of the TSHβ gene, GATA2 transactivates the prepro-TRH gene and that T3-bound TRβ2 interferes with its function, resulting in the negative regulation of this gene.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the [Beta] subunit of TSH (TSH[Beta]). We previously reported that T3-dependent repression of the TSH[Beta] gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived T[alpha] T1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with T[alpha] T1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TR[Beta]2. The inhibition by T3-bound TR[Beta]2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TR[alpha] 1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSH[Beta] gene.

Senescent cells exhibit several typical features, including the senescence-associated secretory phenotype (SASP), promoting the secretion of various inflammatory proteins and small extracellular vesicles (EVs). SASP factors cause chronic inflammation, leading to age-related diseases. Recently, therapeutic strategies targeting senescent cells, known as senolytics, have gained attention; however, noninvasive methods to detect senescent cells in living organisms have not been established. Therefore, the goal of this study was to identify novel senescent markers using small EVs (sEVs). sEVs were isolated from young and senescent fibroblasts using three different methods, including size-exclusion chromatography, affinity column for phosphatidylserine, and immunoprecipitation using antibodies against tetraspanin proteins, followed by mass spectrometry. Principal component analysis revealed that the protein composition of sEVs released from senescent cells was significantly different from that of young cells. Importantly, we identified ATP6V0D1 and RTN4 as novel markers that are frequently upregulated in sEVs from senescent and progeria cells derived from patients with Werner syndrome. Furthermore, these two proteins were significantly enriched in sEVs from the serum of aged mice. This study supports the potential use of senescent markers from sEVs to detect the presence of senescent cells in vivo.