Connexin-32 acts as a downregulator of growth of thyroid gland.

Thyroid epithelial cells communicate through gap junctions formed from connexin (Cx)32, Cx43, and Cx26. We previously reported that reexpression of Cx32 in "gap junction-deficient" FRTL-5 and FRT thyroid cell lines induces a reduction of cell proliferation rate and an activation of expression of cell differentiation. The present study aimed at determining whether Cx32 could exert similar regulatory functions in vivo. We investigated morphological and functional characteristics of thyroid gland of Cx32-deficient mice (Cx32-KO), mice overexpressing Cx32 selectively in the thyroid (Cx32-T+), and Cx32-KO mice with a thyroid-selective Cx32 complementation obtained by crossing Cx32-KO and Cx32-T+ mice. In basal conditions, Cx32-KO mice did not present any detectable thyroid alteration, whereas Cx32-T+ mice showed a thyroid hypoplasia (20% reduction) associated with a slight increase in thyroid functional activity. Under thyrotropin stimulation (following sodium perchlorate treatment), Cx32-KO mice developed a larger goiter (< or =65% increase) than wild-type littermates, whereas Cx32-T+ mice exhibited the same thyroid hyperplasia as wild-type mice. Restoration of Cx32 expression in the thyroid of Cx32-KO mice abrogated the thyroid growth increase related to Cx32 deficiency. All together, these data show that Cx32 acts as a downregulator of growth of thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to TSH-stimulated thyroid cells.

The porcine sodium/iodide symporter gene exhibits an uncommon expression pattern related to the use of alternative splice sites not present in the human or murine species.

The sodium/iodide symporter (NIS) is a membrane protein mediating the active transport of iodide into the thyroid gland. NIS, expressed by human, rat, and mouse thyrocytes, is encoded by a single transcript. We identified NIS mRNA species of 3.5 and 3 kb in porcine thyrocytes. Because porcine thyrocytes in primary culture is a widely used experimental system for thyroid iodide metabolism, we further examined the origin and the function of the porcine NIS (pNIS) transcripts. We generated a porcine thyroid cDNA library from which four different clones, pNIS-D, F, J, and Delta J were isolated. pNIS-D encodes a protein of 643 amino acids highly homologous to the human, rat, and mouse NIS. pNIS-F and J differ from each other and from pNIS-D in their C-terminal part. pNIS-Delta J lacks a six-amino-acid segment within the putative transmembrane domain 10. Transiently expressed in Cos-7 cells, the four pNIS-cDNAs led to the synthesis of proteins targeted at the plasma membrane and conferred perchlorate-sensitive iodide uptake activities to Cos-7 cells, except pNIS-Delta J, which was devoid of activity. PNIS-D probably derives from the 3.5-kb transcript and pNIS-F, J, and Delta J from the 3-kb transcript. The relative abundance of pNIS-D, F, and J transcripts in porcine thyrocytes was about 60%, 35%, and 5%, respectively; the Delta J transcript was not present in detectable amount. By comparing porcine NIS genomic and cDNA sequences, splice donor and acceptor sites accounting for the generation of pNIS-F, J, and Delta J transcripts were identified. None of the combinations of alternative splice sites found in the pig was present in the human, rat or mouse NIS gene. Our data show that porcine NIS gene, contrary to the NIS gene from other species, gives rise to splice variants leading to three active and one inactive NIS proteins.

Thyroid cell proliferation in response to forced expression of gap junction proteins.

Gap junctions are known to play a role in the control of cell proliferation, and connexins (Cx) are considered to be tumor suppressors. However, the effects of Cx on cell proliferation are dependent on the Cx which is expressed and on the cell type under consideration. We previously found that restoration of cell-to-cell communication by stable transfection of two independent thyroid-derived cell lines, FRTL-5 and FRT cells, with the Cx32 gene induced a marked reduction of their proliferation rate. This study aimed i) at determining whether Cx43, which is coexpressed with Cx32 by thyroid epithelial cells, exerts the same action as Cx32 on cell proliferation and ii) at identifying alterations of the cell cycle control system that might account for the Cx32-induced proliferation slowdown in thyrocytes. In contrast with previous data on different epithelial cell types, we report that restoration of intercellular communication in FRTL-5 and FRT cells by stable expression of Cx43 did not modify their proliferation properties. Cell cycle analyses revealed that the Cx32-induced proliferation slow-down was related to a lengthening of the G1 phase. The level of expression of two regulatory proteins of the Cip/Kip cyclin-dependent kinase inhibitor family, p27kip1 and p2cip1, was increased in the two cell lines expressing Cx32. In conclusion, Cx32 and Cx43, physiologically coexpressed by thyrocytes, have a differential impact on thyroid cell proliferation in vitro. The cyclin-dependent kinase inhibitors, p27kip1 and p21cip1 might represent cell cycle effectors relaying the down-regulatory effect of Cx32 on the proliferation of thyroid epithelial cells.