TGF-beta regulation of focal adhesion proteins and motility of premalignant oral lesions via protein phosphatase 1

Abstract

Premalignant oral lesions have a high incidence of recurrence and progression to malignant disease and, although studies have shown the contribution of transforming growth factor β (TGF-β) to cancer progression, none have been conducted with premalignant oral lesion cells to determine the impact of TGF-β in stimulating properties that are characteristic of more invasive cells. The present study focused on TGF-β-modulation of paxillin and the serine/threonine protein phosphatase PP-1, and the impact on cellular motility. These studies show that TGF-β stimulates premalignant lesion cell motility and up regulates expression of paxillin, as well as its co-localization with PP-1, while concurrently diminishing the level of paxillin serine phosphorylation. The TGF-β-mediated up regulation of paxillin and co-localization with actin, as well as the TGF-β-stimulated motility of premalignant lesion cells, were all blocked by inhibiting PP-1, indicating their dependence on PP-1 activity. These studies suggest interplay between TGF-β and PP-1 in promoting a more malignant phenotype in premalignant oral lesion cells.

Figure 1

Analyses of paxillin immunoprecipitates for levels of paxillin, actin and serine-phosphorylated paxillin in TGF-β-treated cells. Top panel: Representative Western blots following treatment of premalignant oral lesion cells with 5 ng/ml TGF-β for 24 hours. Paxillin immunoprecipitates from these treated cells were separated and blotted with antibodies to paxillin, actin, and phosphoserine. Lower panel: Mean densitometric scans of 3 separate immunoprecipitates and immunoblots from TGF-β-treated premalignant lesion cells. Data shown are means±SD.

Figure 2

Analyses of paxillin immunoprecipitates for levels of paxillin and co-precipitated PP-1 in TGF-β-treated cells. Top panel: Representative Western blots following treatment of premalignant oral lesion cells with TGF-β for 24 hours. Paxillin immunoprecipitates from these treated cells were separated and blotted with anti-paxillin and antibody to the catalytic subunit of PP-1. Lower panel: Mean densitometric scans of 3 separate immunoprecipitates and immunoblots from TGF-β-treated premalignant lesion cells. Data shown are means±SD.

Figure 3

TGF-β increases PP-1 enzyme activity in premalignant oral lesion cells. Premalignant lesion cells were treated with 5 ng/ml TGF-β and, after various time increments, lysed and used to measure PP-1 enzyme activity. Data shown are means of triplicate assays + SEM.

Figure 4

TGF-β-mediated increases in paxillin protein levels and co precipitation of actin are dependent on PP-1. Top panel: Representative Western blots following treatment of premalignant oral lesion cells with 5 ng/ml TGF-β and/or 500 nM tautomycetin (TMC). Paxillin immunoprecipitates were blotted with anti-paxillin and anti-actin antibodies. Bottom panel: Mean densitometric scans of 3 separate immunoprecipitates and immunoblots from TGF-β-treated premalignant lesion cells. Data shown are means±SD.

Figure 5

TGF-β–mediated premalignant lesion cell migration is PP-1 dependent. The migration of premalignant lesion cells from the upper compartment to the lower compartment of a transwell migration chamber was assessed in the presence of various doses of TGF-β with or without 500 nM tautomycetin (TMC) to inhibit PP-1 activity. Migration is expressed as densitometric units that are proportional to cell numbers. Error bars represent SEM for triplicate measurements in 3 separate experiments.