Differential control of connexin-32 and connexin-43 expression in thyroid epithelial cells: evidence for a direct relationship between connexin-32 expression and histiotypic morphogenesis.
Thyroid epithelial cells cultured either as a monolayer or in the form of follicles, rapidly reconstitute functional gap junctions (Gj). We previously reported that the thyroid Gj gating is regulated by TSH. We have now performed molecular analyses of Gj proteins 1) to detect the connexin(s) (Cx) that is expressed in thyroid epithelial cells, 2) to determine whether the expression of Cx is hormonally regulated, and 3) to analyze the relationship between Cx expression and histiotypic morphogenesis, i.e. folliculogenesis. Studies were carried out on thyrocytes freshly isolated from the gland and on corresponding thyrocytes after 1-7 days in culture as monolayers or in the form of reconstituted follicles. The Cx gene transcription products were analyzed by Northern blot using specific complementary DNA probes for Cx26, Cx32, and Cx43. Cx proteins were identified and estimated by Western blot and indirect immunofluorescence using polyclonal antipeptide antibodies. Cx32 and Cx43 proteins and their corresponding messenger RNA (mRNA) were detected in thyrocytes freshly isolated from the gland. Thyrocytes contained a high amount of the 1.6-kilobase Cx32 mRNA and only traces of the 3-kilobase Cx43 transcript. No Cx26 transcripts could be detected. Thyrocytes cultured at a density of 0.2-0.5 x 10(6) cells/cm2 in the absence of TSH formed monolayers. Surprisingly, monolayer cells lost Cx32 protein within 24 h, and their Cx32 mRNA content decreased from high to barely detectable levels; Cx32 protein was no longer detected throughout the 1-week culture period. On the contrary, Cx43 mRNA and Cx43 protein rapidly increased in monolayer cells to reach very high levels within 2-4 days. Thyrocytes cultured at the same density, but in the presence of TSH also rapidly lost Cx32, but as soon as they reorganized into follicular structures, reexpressed Cx32 at a level (in terms of protein and mRNA) comparable to that found in cells freshly extracted from the gland. As observed for cell monolayers, reconstituted follicles overexpressed Cx43. The Cx43 protein and Cx43 mRNA contents of cultured thyrocytes were 20- to 50-fold higher than those found in isolated thyrocytes at the outset of culture. When thyrocytes were cultured with TSH, but at a low density (< 0.2 x 10(6) cells/cm2) to prevent follicle formation, a TSH-dependent increase in Cx43 was observed in monolayer cells. However, TSH did not cause any reexpression of Cx32.(ABSTRACT TRUNCATED AT 400 WORDS)
Munari-Silem
Y
Y
Institut National de la Santé et de la Recherche Médicale U-369, Faculté de Médecine A. Carrel, Lyon, France.
Guerrier
A
A
Fromaget
C
C
Rabilloud
R
R
Gros
D
D
Rousset
B
B
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D018031:Connexin 43 / Q000032:analysis / Q000235:genetics*; D017630:Connexins / Q000032:analysis / Q000235:genetics*; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D007700:Kinetics; D009024:Morphogenesis / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine; D013961:Thyroid Gland / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology; D000097002:Gap Junction beta-1 Protein
D018031:Connexin 43; D017630:Connexins; D012333:RNA, Messenger; D013972:Thyrotropin; D000097002:Gap Junction beta-1 Protein