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Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

ABSTRACT: To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA).Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 family). Proteins were extracted for western blot analysis of p57 and p15. For RNA interference, primary cultured human limbal epithelial cells were transfected with annealed double-stranded siRNA (67 nM) specific for p57, p15, or siRNA-Fluorescein (siRNA-F; as a negative control) followed by treatment with TGF-beta1 or TGF-beta2 at 1 ng/ml. P57 and p15 were quantitatively detected by real-time PCR and western blot; and immunolocalized by immunofluorescent staining. The effects of TGF-beta1 or TGF-beta2 on cell proliferation were evaluated by BrdU incorporation and MTT assay.TGF-beta1 or TGF-beta2 significantly inhibited primary cultured human limbal epithelial cell proliferation measured by BrdU incorporation and MTT assay. TGF-beta1 or TGF-beta2 upregulated the expression of p57 and p15 mRNA and protein, but did not effect the expression of p19, p21, or p27. The siRNA transfection efficiency of these cells was 75% and no cellular toxicity was observed by 24 h. The TGF-beta1 or TGF-beta2 stimulated expression of p57 and p15 mRNA were markedly blocked by siRNA-p57 or siRNA-p15, respectively, but not by siRNA-F. The TGF-beta1 or TGF-beta2 suppression of epithelial proliferation measured by BrdU incorporation and MTT generation was increased to near normal levels by siRNA-p57 or siRNA-p15. Western blot and immunofluorescent staining showed that levels of p57 and p15 proteins were equally reduced in the cytoplasm and nucleus.These findings demonstrate that TGF-beta1 and/or TGF-beta2 inhibit proliferation of primary cultured human limbal epithelial cells and that p57 and p15 play roles in this process.

SUBMITTER: Chen Z

PROVIDER: S-EPMC2906388 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

Chen Zhuo Z Li De-quan DQ Tong Louis L Stewart Paul P Chu Claire C Pflugfelder Stephen C SC

Molecular vision 20060823

<h4>Purpose</h4>To evaluate the role of cyclin-dependent kinase inhibitors p57 and p15 in transforming growth factor (TGF)-beta1 or TGF-beta2 inhibited proliferation of primary cultured human limbal epithelial cells using short interfering RNA (siRNA).<h4>Methods</h4>Primary cultured human limbal epithelial cells were treated with TGF-beta1 or TGF-beta2 for 6 and 24 h, and total RNA extracted for RT-PCR and real-time PCR using primers for p21, p27, and p57 (CipP/Kip family) and p15 and p19 (INK4 ...[more]

PMID: 16943770

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Project description:PurposeContinuous culture of limbal epithelial stem cells (LSCs) slows down proliferation, which inevitably results in differentiation. Transforming growth factor-beta (TGFβ)-assisted epithelial-mesenchymal transition (EMT) is often found in the late stage of LSC culture. Thus, EMT is proposed to be part of the mechanism responsible for the loss of LSCs in culture. To explore the regulation mechanism of EMT, we investigated the early stage culture for factor(s) that may potentially prevent EMT.MethodsLSCs from the corneal limbus region of rabbits were isolated and expanded to confluence in culture (P0), and then serial passage of these LSCs (P1 to P3) was performed. EMT in LSCs was induced with TGFβ1, and the corresponding EMT signaling was confirmed with Smad2/3 phosphorylation. The expression of mesenchymal markers, including alpha-smooth muscle actin (α-SMA) and vimentin, was determined with western blot analysis. Proteins extracted from different passaged cells were also subjected to western blot analysis of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3 as well as Smad7, the main negative regulator of TGFβ signaling. The mitogenic response was measured with the bromodeoxyuridine (BrdU) labeling index and real-time PCR using primers for Ki67. N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, and Jagged-1 Notch ligand were used to block and activate Notch signaling, respectively, and their efficacy was evaluated by determining the expression of Hes1, a Notch signaling target.ResultsMesenchymal marker induction and growth arrest were found in the TGFβ1-treated P1 cells, and the changes were less significant in the TGFβ1-treated P0 cells. Western blot analysis confirmed that the expressed levels of TGFβ signaling components, including TGFβ1, TGFβ receptor I/II, and Smad2/3, were relatively stable with passages. In contrast, the expression of Hes1 and Smad7 markedly decreased after the first passage, and with each passage, the levels diminished even further. Hes1 and Smad7 were expressed only in the limbal epithelium and not in the corneal epithelium. DAPT effectively blocked the expression of Hes1. DAPT also dose-dependently suppressed Smad7 expression in P0 cells, which was associated with the susceptibility of P0 cells to TGFβ1-induced Smad2/3 phosphorylation, EMT formation, and growth arrest. Reciprocally, Jagged-1 upregulated Smad7 expression in LSCs against TGFβ signaling.ConclusionsThese findings indicate that Smad7 plays a crucial role in antagonizing EMT induced by TGFβ signaling and support our proposition that Smad7 is a Notch signaling target in LSCs, and may mediate the Notch function in preventing the occurrence of EMT.

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Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

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Targeted inhibition of p57 and p15 blocks transforming growth factor beta-inhibited proliferation of primary cultured human limbal epithelial cells.

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