A germline mutation (A339V) in thyroid transcription factor-1 (TITF-1/NKX2.1) in patients with multinodular goiter and papillary thyroid carcinoma.

BACKGROUND: The genetic factors that determine the risk of papillary thyroid carcinoma (PTC) among patients with multinodular goiter (MNG) remain undefined. Because thyroid transcription factor-1 (TTF-1) is important to thyroid development, we evaluated whether the gene that encodes it, TITF-1/NKX2.1, is a genetic determinant of MNG/PTC predisposition. METHODS: Twenty unrelated PTC patients with a history of MNG (MNG/PTC), 284 PTC patients without a history of MNG (PTC), and 349 healthy control subjects were screened for germline mutation(s) in TITF-1/NKX2.1 by sequencing of amplified DNA from blood. The effects of the mutation on the growth and differentiation of thyroid cells were demonstrated by ectopic expression of wild-type (WT) and mutant proteins in PCCL3 normal rat thyroid cells, followed by tests of cell proliferation, activation of cell growth pathways, and transcription of TTF-1 target genes. All statistical tests were two-sided. RESULTS: A missense mutation (1016C>T) was identified in TITF-1/NKX2.1 that led to a mutant TTF-1 protein (A339V) in four of the 20 MNG/PTC patients (20%). These patients developed substantially more advanced tumors than MNG/PTC or PTC patients without the mutation (P = .022, Fisher exact test). Notably, this germline mutation was dominantly inherited in two families, with some members bearing the mutation affected with MNG, associated with either PTC or colon cancer. The mutation encoding the A339V substitution was not found among the 349 healthy control subjects nor among the 284 PTC patients who had no history of MNG. Overexpression of A339V TTF-1 in PCCL3 cells, as compared with overexpression of WT TTF-1, was associated with increased cell proliferation including thyrotropin-independent growth (average A339V proliferation rate = 134.27%, WT rate = 104.43%, difference = 34.3%, 95% confidence interval = 12.0% to 47.7%, P = .010), enhanced STAT3 activation, and impaired transcription of the thyroid-specific genes Tg, TSH-R, and Pax-8. CONCLUSION: This is the first germline mutation identified in MNG/PTC patients. It could contribute to predisposition for MNG and/or PTC and to the pathogenesis of PTC.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis.

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

Prokineticin-1 (Prok-1) works coordinately with glial cell line-derived neurotrophic factor (GDNF) to mediate proliferation and differentiation of enteric neural crest cells.

Enteric neural crest cells (NCC) are multipotent progenitors which give rise to neurons and glia of the enteric nervous system (ENS) during fetal development. Glial cell line-derived neurotrophic factor (GDNF)/RET receptor tyrosine kinase (Ret) signaling is indispensable for their survival, migration and differentiation. Using microarray analysis and isolated NCCs, we found that 45 genes were differentially expressed after GDNF treatment (16 h), 29 of them were up-regulated including 8 previously undescribed genes. Prokineticin receptor 1 (PK-R1), a receptor for Prokineticins (Prok), was identified in our screen and shown to be consistently up-regulated by GDNF in enteric NCCs. Further, PK-R1 was persistently expressed at a lower level in the enteric ganglions of the c-Ret deficient mice when compared to that of the wild-type littermates. Subsequent functional analysis showed that GDNF potentiated the proliferative and differentiation effects of Prok-1 by up-regulating PK-R1 expression in enteric NCCs. In addition, expression analysis and gene knock-down experiments indicated that Prok-1 and GDNF signalings shared some common downstream targets. More importantly, Prok-1 could induce both proliferation and expression of differentiation markers of c-Ret deficient NCCs, suggesting that Prok-1 may also provide a complementary pathway to GDNF signaling. Taken together, these findings provide evidence that Prok-1 crosstalks with GDNF/Ret signaling and probably provides an additional layer of signaling refinement to maintain proliferation and differentiation of enteric NCCs.

Induction of functional MT1 and MT2 isoforms by calcium in anaplastic thyroid carcinoma cells.

Metallothionein (MT) expression in carcinogenesis of thyrocytes is unknown. We demonstrated that cadmium induced transcription of all functional MT-1 and MT-2 isoforms and promoted the cell cycle from the G1 to the S phase in thyroid cancer cells, which can be suppressed by the ERK inhibitor. Cadmium exposure stimulated intracellular calcium and the phosphorylation of ERK1/2. Therefore, a common pathway initiated by a rapid rise in calcium and followed by calcium-mediated activation of ERK is involved in the transcriptional induction of functional MT1 and MT2 isoforms and in the progression of the cell cycle in thyroid cancer cells exposed to cadmium.