Transforming Growth Factor Beta 1 Binding and Receptor Kinetics in Fetal Mouse Lung Fibroblasts

Abstract

TGFbeta1 inhibits fetal lung maturation in vitro. As TGFbeta1 is present in fetal lung, mechanisms must exist to overcome this inhibition and allow late gestation maturation to progress. We studied the ontogeny of TGFbeta1 binding, and TGFbeta receptor kinetics and subtypes in primary cultures of fetal mouse lung fibroblasts from Day 16 to Day 18 of gestation. TGFbeta1 specific binding in fetal lung fibroblasts declined with advancing gestation. The decrease occurred earlier, and was more pronounced in female fibroblasts (50% decrease) than in the male fibroblasts (29% decrease). Dihydrotestosterone treatment of Day 18 female fibroblasts resulted in a dose-dependent increase in TGFbeta1 binding. Scatchard analysis revealed a decline in receptor number with advancing gestation (Day 16 female Bmax: 7.3 x 10(-16); Day 18 female Bmax: 5.5 x 10[-16]) whereas binding affinities remained constant. Affinity labeling followed by chemical cross-linking and autoradiography showed the three known TGFbeta receptor subtypes at both Days 16 and 18 of gestation. The relative abundance of nonsignaling Type III receptors in comparison to signaling Type II and Type I receptors was increased at Day 18 versus Day 16. Incorporation of [3H]thymidine into DNA after treatment with TGFbeta1 changed from Day 16 to Day 18, consistent with changes previously reported between fetal and adult lung fibroblasts. We conclude that as fetal mouse lung maturation progresses, TGFbeta receptor number decreases in fibroblasts, the relative proportion of nonsignaling versus signaling receptor types increases, and the response to TGFbeta1 stimulation changes. These changes may contribute to overcoming TGFbeta1 inhibition of lung maturation.

Androgen Regulation of Signaling Pathways in Late Fetal Mouse Lung Development1

“…Ϯ 1145 dpm/nmol DNA; male cells, 3894 Ϯ 521 dpm/nmol DNA; P Ͻ 0.05), similar to results we have previously reported (27). DHT exposure also caused a significant increase over baseline thymidine incorporation in female fibroblasts (4187 Ϯ 1055 dpm/nmol DNA; P Ͻ 0.05), and a mild decrease in male fibroblasts (1872 Ϯ 436 dpm/ nmol DNA).…”

“…This dose of DHT was chosen because a similar dose was used in mice to study androgen effects on fetal sexual differentiation (32) and to document androgen effects on the fetal lung (4,8). Fibroblast cell cultures were prepared using procedures previously described (20,22,27,34). The uterus was removed under sterile conditions by laparotomy and kept on ice.…”

“…The assay was performed as previously described by us with modifications (27). Cultures (at 90 -95% confluence) were washed with and then incubated in dissociation buffer (DMEM containing 25 mm HEPES and 0.1% BSA) for 2 h at 37 C to allow for dissociation or internalization of endogenous bound TGF␤1.…”

“…For example, androgen inhibition of fibroblast-type II cell communication has been reversed with antibodies to TGF␤1 (23). All of these events were accompanied by an alteration in the relative proportions of receptor subtypes and an altered proliferative response to TGF␤1 stimulation (27). We previously reported that TGF␤-R binding and total receptor number decrease in late gestation fetal lung fibroblasts.…”

“…The effect of DHT on EGF-R and TGF␤-R activity was measured in late gestation when differences in maturation have resolved (28,29), and the baseline activities of these receptors are similar in both sexes (22,27). We proposed that the inhibitory effect of androgens on the development of fibroblast-type II cell communication is mediated by an alteration of the developmental changes in these receptors.…”