TGFbeta2 activation status during cardiac morphogenesis.
Transforming growth factor beta (TGFbeta) is secreted as a biologically inactive complex by many cell types in vitro, but little is known of TGFbeta's activation status in vivo. This study examined the in vivo expression of active and total (active + acid-activatable) TGFbeta2 in embryonic chicken hearts during cardiac morphogenesis (Hamburger-Hamilton stage 10-24). The concentration of TGFbeta2 was measured by an enzyme-linked immunoassay that recognized active TGFbeta2. Whole heart homogenates were either left untreated to measure active TGFbeta2 or treated with acid before assay to measure total (active + acid-activatable) TGFbeta2. Total TGFbeta2 concentration increased more than 16-fold between stage 10/11 and stage 24. Active TGFbeta2 concentration was highest at stage 14/15, but overall remained relatively constant varying at most by 2.8-fold. When expressed relative to total TGFbeta2, the amount of active TGFbeta2 progressively declined from 70% in stage 10/11 hearts to 7% in stage 24 hearts. The distribution of active and total TGFbeta2 was examined by immunostaining with an antibody against active TGFbeta2. Before immunostaining, sections were either treated with acid or left untreated to determine the distribution of total and active TGFbeta2, respectively. Active TGFbeta2 immunostaining was first detected in the endothelium, myocardium, and cardiac jelly of stage 14 hearts. Acid treatment had no effect on the distribution or intensity of immunostaining at this stage. Faint, active TGFbeta2 immunostaining was restricted to the ventricular myocardium in stage 18 hearts. Acid treatment resulted in a marked increase in staining intensity in the ventricle, but no staining was observed in the atrium or outflow tract. In stage 24 hearts, faint active TGFbeta2 staining was detected in the ventricle before acid treatment. After acid treatment, patches of intense punctate stain were found in all regions of the embryonic heart. Increases in TGFbeta2 concentration and immunostaining intensity after acidification suggest that a significant amount of TGFbeta2 is in the latent form. Stage-dependent differences in activation status suggest that activation may be a developmentally regulated process in the chick heart and support the notion that activation is an important step in regulating TGFbeta actions in vivo.
McCormick
K M
KM
Department of Physical Therapy, Exercise and Nutrition Sciences, University at Buffalo, State University of New York, Buffalo, New York 14214, USA. kmmccorm@buffalo.edu
D000818:Animals; D015153:Blotting, Western; D002642:Chick Embryo; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D006321:Heart / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis; D013997:Time Factors; D014018:Tissue Distribution; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000378:metabolism*; D053781:Transforming Growth Factor beta2
D017395:Plasminogen Activator Inhibitor 1; D016212:Transforming Growth Factor beta; D053781:Transforming Growth Factor beta2; D008156:Luciferases