Abstract
Growth activation of quiescent cells leads to enhanced low density lipoprotein (LDL) receptor expression at the cell surface. To determine the basis for this stimulated receptor activity, we measured LDL receptor activity, changes in receptor protein mass, and mRNA abundance in quiescent and growth-activated cultured human skin fibroblasts. Growth activation, using insulin or platelet-derived growth factor, led to dose-dependent increases in cellular LDL receptor mRNA level (average 5.2-fold increase at 10 ng/ml platelet-derived growth factor, 4.1-fold increase at 58 ng/ml insulin) and cell surface expression (average 3.5-fold increase at 10 ng/ml platelet-derived growth factor, 2.5-fold increase at 58 ng/ml insulin). Increased LDL receptor mRNA levels could be detected as early as 2 h after addition of growth factor (3.2-fold), whereas increased levels of LDL receptor binding and mass were not detected until after 4-8 h. Growth activation led to induction of LDL receptor gene transcription, led to induction of LDL receptor gene transcription, and the increase of LDL receptor mRNA produced by addition of growth factor was completely prevented by actinomycin D. These observations indicate that growth-related activation of the LDL receptor pathway is accounted for, primarily, by growth-activated enhancement of LDL receptor gene transcription.
Highlights
The low density lipoprotein (LDL)’ receptor is a widely distributed cellular endocytic receptor that functions to internalize cholesterol-containing LDL particles (1, 2)
Growth activation of quiescent cells with serum has been shown to positively modulatesynthesis of certainproteins by modulating cognate mRNA species, whereas other proteins are stimulatedonly at the translational level (15)
We report that growth activation of quiescent human skin fibroblasts leads to enhanced LDL receptor gene transcription
Summary
Cell Culture-Human skin fibroblasts were grown from explants of penile foreskin as previously described (16). Fibroblasts were scraped into a buffer containing 10 incorporated into DNA per milligram of cell protein) and LDL receptor expression in cultured fibroblasts (Fig. lA). Nitrocellulose was trations used, actsasa mitogen for culturedhumanskin fibroblasts This observation is in accord with observations by Conover et al (32), demonstrating increased human skin fibroblast thymidine incorporation and cell number in response to similar insulin concentrations. From 0 to 58 ng/ml insulin containing 0.4 ml of '"I-protein A (Du Pont, 133 pCi/ml, 8.3 pCi/ there is a 4.5-fold increase in LDL receptor mRNA abundance mg) for 2 h at room temperature. 50, 25, 12.5, and 6.25pgof cell extract protein was applied to nitrocellulose paper using a slot blot apparatus and thefilters were processed as described but a2.5-fold increase in LDL receptor cell surface expression.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
