Abstract
Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.
Highlights
Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions
The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells
The low level of coupling observed in FRT cells could be either related to the presence of a low number of Gap junctions (GJ) channels composed of Cx32, Cx43, or Cx26 expressed in minute amounts or dependent on channels composed of another Cx than those normally expressed in thyroid cells
Summary
Expression Vector—The 1.5-kb pCx32 cDNA (containing the entire coding region) was isolated from the pGEM-3 plasmid FRT cells were incubated for 3– 4 h with calcium phosphate in the presence of 20 –30 g of pSVK3-Cx32 and 2–3 g of pCMV-neo and subjected to a 1-min glycerol shock. For FRTL-5 cells, the incubation in the presence of calcium phosphate was reduced to 1 h, and cells were subjected to a 3-min glycerol shock For both cell types, 48 h after the outset of transfection, the neomycin analogue, G418 (Life Technologies, Inc.), was added to the medium at a concentration of 0.4 mg/ml. The rat Pax-8 cDNA fragment (0.3 kbp), corresponding to the paired domain fragment [19], was extracted from the C27 B2xx plasmid by EcoRI and HindIII restriction site digestion. Cells were dissociated by mild trypsinization, and the number of cells/dish was counted under an inverted microscope equipped with phase-contrast optics using an hemocytometer
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