Growth state-dependent regulation of plasminogen activator inhibitor type-1 gene expression during epithelial cell stimulation by serum and transforming growth factor-?1

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Abstract
Transcription of the plasminogen activator inhibitor type‐1 (PAI‐1) gene appears to be growth state regulated in several cell types (e.g., Ryan and Higgins, 1993, J Cell Physiol 155:376–384; Mu et al., 1998, J Cell Physiol 174:90–98). Transit of serum‐stimulated normal rat kidney (NRK) epthelial cells through the first division cycle after release from quiescence (G0) provided a model system to assess the kinetics and mechanisms underlying PAI‐1 expression in a growth “activated” phenotype. PAI‐1 mRNA transcripts increased by more than 20‐fold during the G0→G1 transition; induced expression had immediate‐early response characteristics and abruptly declined prior to the onset of DNA synthesis. Transcriptional activity of the PAI‐1 gene paralleled the steady‐state mRNA abundance profile during this first synchronized growth cycle after release from quiescence. Although PAI‐1 mRNA levels were up‐regulated (approximately threefold) upon exposure to several different growth factors, neutralizing antibodies to transforming growth factor‐β1 (TGF‐β1) effectively attenuated the more than ninefold serum‐associated PAI‐1 inductive response by more than 70% (at both the mRNA transcript and protein levels). Similar to the metabolic requirements for serum‐mediated PAI‐1 transcription, PAI‐1 induction upon addition of TGF‐β1 to quiescent NRK cell cultures was actinomycin D sensitive and resistant to cyclohexamide and puromycin, suggesting a primary mode of transcript control. The response to protein synthesis inhibitors, however, was complex. While cyclohexamide appeared to stabilize, or at least maintain, fetal bovine serum (FBS)‐ or TGF‐β1‐stimulated PAI‐1 mRNA levels, puromycin had no such affect. The amplitude and duration of induced PAI‐1 expression were the same in either the presence or absence of puromycin. Cyclohexamide when used alone (i.e., in non‐FBS‐ or TGF‐β1‐treated cultures), moreover, effectively stimulated PAI‐1 induction whereas puromycin was ineffective. Although TGF‐β1 was not a complete mitogen in the NRK cell system, incubation of quiescent renal cell cultures with TGF‐β1, prior to serum stimulation, resulted in a 10‐ to 12‐fold increase in PAI‐1 expression coincident with exit out of G0. These data support a model in which PAI‐1 gene expression is closely associated with creation of the growth‐activated state and that cell cycle controls appear to be superimposed on the time course of the serum‐induced expression of the PAI‐1 gene. J. Cell. Physiol. 181:96–106, 1999.

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