Caveolae are negative regulators of transforming growth factor-beta1 signaling in ureteral smooth muscle cells.
OBJECTIVE
The mechanisms underlying ureteral cell regulation are largely unknown. Previous studies have identified lipid rafts/caveolae as regulators of growth stimulatory signals in ureteral smooth muscle cells (USMCs). In this study we determined whether growth inhibitory signaling by transforming growth factor-beta1 (TGF-beta1) is also regulated by caveolae in USMC.


METHODS
Expression of components of the TGF-beta1 signaling axis in USMCs was determined by immunoblot and mRNA analyses. Growth regulatory activity of TGF-beta1 was assessed by H-thymidine incorporation. In select experiments caveolae were disrupted reversibly by cholesterol depletion and replenishment prior to TGF-beta1 treatment. TGF-beta1-responsive gene expression was evaluated using the TGF-beta1 responsive promoter-reporter construct 3TP-Lux.


RESULTS
USMCs expressed TGF-beta1, types I and II TGF-beta1 receptors, and the effector Smad-2. TGF-beta1 potently inhibited DNA synthesis in USMCs (IC50 60 pM). TGF-beta1 mediated DNA synthesis inhibition was potentiated following the disruption of caveolae by cholesterol depletion. This effect was reversible with membrane cholesterol restoration. TGF-beta1 stimulated gene activity was augmented by caveolae disruption, while caveolae reformation returned promoter activity to baseline levels.


CONCLUSIONS
TGF-beta1 is a potent growth inhibitor of USMCs and its activity can be enhanced by caveolae ablation. These findings suggest a role for TGF-beta1 in the growth regulation of normal ureteral cells and implicate caveolar membrane domains in the negative regulation of TGF-beta1 signaling. These studies may be relevant to ureteral pathologies that are characterized by smooth muscle dysplasia.
Stehr
Maximilian
M
Department of Urology (Urological Diseases Research Center), Children's Hospital Boston, Massachusetts 02115, USA.
Estrada
Carlos R
CR
Khoury
Joseph
J
Danciu
Theodora E
TE
Sullivan
Maryrose P
MP
Peters
Craig A
CA
Solomon
Keith R
KR
Freeman
Michael R
MR
Adam
Rosalyn M
RM
D000818:Animals; D021941:Caveolae / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology* / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014513:Ureter / Q000166:cytology* / Q000502:physiology*
C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1