Abstract
The integrin family is composed of a large number of heterodimers, each one mediating distinct interactions with extracellular matrix and/or cell surface ligands. The expression of integrins appears to be tightly regulated in vivo, but the mechanisms by which cells control the formation and surface expression of specific pairs of subunits have not been well characterized. Two integrin subunits, the alpha subunit alpha v, and the beta subunit beta 1, could pose special problems in regulation because of their capacity to associate with multiple partners. In the present study, we have examined the effects of the cytokine transforming growth factor beta 1 (TGF-beta 1) on the expression of alpha v- and beta 1-containing integrins in primary cultures of guinea pig airway epithelial cells, e.g. cells that we have previously found to express multiple potential partners for both alpha v and beta 1. TGF-beta 1 increased the surface expression of both alpha v- and beta 1-containing heterodimers after periods of stimulation from 24 to 72 h. These increases in surface expression were associated with significant increases in the concentrations of mRNA encoding each of the partners of alpha v and beta 1, but with only minimal increases in mRNA encoding alpha v and beta 1 themselves. Airway epithelial cells metabolically labeled with [35S]methionine during stimulation with TGF-beta 1 demonstrated only a minimal increase in the synthesis of new alpha v protein at a time when synthesis of alpha v's beta subunit partners and surface expression of alpha v-containing heterodimers were dramatically increased. These data suggest that, at least in some cells, promiscuous integrin subunits (both alpha and beta) may normally be synthesized in excess. Thus, the surface expression of specific integrin heterodimers can be regulated primarily through regulation of the synthesis of the specific partners of these subunits.
Highlights
The integrin family is composed of a large number linked
We have examined the effects of the cytokine transforming growth factor81 (TGF-81) on the expression of av- and 81-containing integrins in primary cultures of guinea pig airway epithelial cells, e.g. cells that we have previously found to express multiple potential partners for both av and
Immunoprecipitation of au-containing Integrins-We have previously reported that guinea pig tracheal epithelial cells express mRNA encoding av and at least three potential p subunit partners: p l, p3, and p6 (15)
Summary
Antibodies, and Reagents-Male Hartley outbred guinea pigs weighing 500-1000 g were obtained from a cesarean-originated, barrier-sustained colony (Charles River Breeding Laboratories). A rabbit polyclonal antiserum raised against the cytoplasmic domain of the integrin subunit av (18)was a gift from L. Suspended tracheal epithelial cells were radioiodinatedusing 0.3 mCi of sodium [lZ5I] iodide per filter support in thesame buffer supplemented with 1mg/ ml of lactoperoxidase and 214 pl/ml of glucose oxidase. Metabolic Labeling-Confluent filters of airway epithelial cells were incubated with either serum-free medium or medium supplemented with 250 p~ TGF-61 (R & DSystems Inc., Minneapolis, MN). At the end of the 30-min pulse, cells were washed three times and lysed in immunoprecipitation buffer. Equal volume aliquots of lysates of metabolically labeled cells were boiled in 1%SDS to dissociate subunits, diluted in immunoprecipitation buffer and immunoprecipitated as above, except that thebuffer was supplemented to 400 mM NaCl during the four washes of the immunoprecipitated samples, and the gels were fluorographed prior to autoradiography. Densitometry values obtained for each integrin subunit probe were normalized based on the values obtained when the same filters were probed for 0-actin
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