TGF-beta1 mediates glucose-evoked up-regulation of connexin-43 cell-to-cell communication in HCD-cells.
OBJECTIVE
In the current study we examined if the multifunctional cytokine TGF-beta1 mediated glucose-evoked increases in connexin-43(Cx43)-mediated intercellular communication in cells of the human collecting duct (HCD).


METHODS
RT-PCR and western blot analysis were used to confirm mRNA and protein expression of TGF-beta1 and Cx43 in HCD-cells. The effect of TGF-beta1 and high glucose (25 mM) on Cx43 protein expression, cytoskeletal organisation and cell-cell communication was determined in the presence/absence of TGF-beta1 specific immuno-neutralising antibodies. Functional cell-cell communication was determined using Ca2+-microfluorimetry.


RESULTS
At 24 hrs, high glucose (25 mM) significantly increased Cx43 mRNA and protein expression. Changes were mimicked by TGF-beta1 (2 ng/ml) at low glucose (5 mM). Both high glucose and TGF-beta1 mediated changes were completely reversed by a pan-specific immuno-neutralising antibody to TGF-beta. Furthermore, high glucose-evoked changes were inhibited by a TGF-beta1-specific monoclonal antibody. Mannitol (25 mM), an osmotic control for high glucose, failed to alter Cx43 expression. TGF-beta1 evoked changes in Cx43 expression were biphasic. An early (4-8 hr) transient decrease in expression was followed by an increase in protein expression (12-24 hr). The decrease in Cx43 expression was paralleled by a transient reorganisation of the actin cytoskeleton, whilst increased Cx43 expression at 24 hrs coincided with a TGF-beta1 specific increase in touch-evoked transmission of Ca2+-signals between coupled cells.


CONCLUSIONS
High glucose evoked a TGF-beta1 mediated increase in Cx43 expression and gap-junction mediated cell-cell communication in HCD-cells. These changes may maintain epithelial integrity of the collecting duct following hyperglycaemic assault as observed in diabetes.
Hills
Claire E
CE
Department of Infection, Immunity and Inflammation, MSB building, University of Leicester.
Bland
Rosemary
R
Bennett
Jeanette
J
Ronco
Pierre M
PM
Squires
Paul E
PE
D000199:Actins / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D007685:Kidney Tubules, Collecting / Q000166:cytology* / Q000378:metabolism*; D010590:Phalloidine / Q000378:metabolism; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D013539:Simian virus 40 / Q000502:physiology; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D000199:Actins; D018031:Connexin 43; D005456:Fluorescent Dyes; D011506:Proteins; D012333:RNA, Messenger; D012235:Rhodamines; D053773:Transforming Growth Factor beta1; D010590:Phalloidine; C009434:tetramethylrhodamine isothiocyanate; D005947:Glucose