Keratinocyte growth factor-2 stimulates P-glycoprotein expression and function in intestinal epithelial cells

Abstract

Intestinal P-glycoprotein (Pgp/multidrug resistance 1), encoded by the ATP-binding cassette B1 gene, is primarily involved in the transepithelial efflux of toxic metabolites and xenobiotics from the mucosa into the gut lumen. Reduced Pgp function and expression has been shown to be associated with intestinal inflammatory disorders. Keratinocyte growth factor-2 (KGF2) has emerged as a potential target for modulation of intestinal inflammation and maintenance of gut mucosal integrity. Whether KGF2 directly regulates Pgp in the human intestine is not known. Therefore, the present studies were undertaken to determine the modulation of Pgp by KGF2 using Caco-2 cells. Short-term treatment of Caco-2 cells with KGF2 (10 ng/ml, 1 h) increased Pgp activity (~2-fold, P < 0.05) as measured by verapamil-sensitive [(3)H]digoxin flux. This increase in Pgp function was associated with an increase in surface Pgp levels. The specific fibroblast growth factor receptor (FGFR) antagonist PD-161570 blocked the KGF2-mediated increase in Pgp activity. Inhibition of the mitogen-activated protein kinase (MAPK) pathway by PD-98059 attenuated the stimulatory effects of KGF2 on Pgp activity. Small-interfering RNA knockdown of Erk1/2 MAPK blocked the increase in surface Pgp levels by KGF2. Long-term treatment with KGF2 (10 ng/ml, 24 h) also significantly increased PgP activity, mRNA, protein expression, and promoter activity. The long-term effects of KGF2 on Pgp promoter activity were also blocked by the FGFR antagonist and mediated by the Erk1/2 MAPK pathway. In conclusion, our findings define the posttranslational and transcriptional mechanisms underlying stimulation of Pgp function and expression by KGF2 that may contribute to the beneficial effects of KGF2 in intestinal inflammatory disorders.

Lactobacillus acidophilusstimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells

“…Caco-2 cells were grown routinely in 75-cm 2 plastic flasks in minimum essential medium (pH 7.2) supplemented with high-glucose 20% FBS, 20 mM HEPES, 100 IU/ml penicillin, and 100 g/ml streptomycin in 5% CO 2-95% O2 at 37°C (48,49). Cells at passages 25-45 were used for these studies.…”

“…Cells at passages 25-45 were used for these studies. For promoter studies, Caco-2 cells were transiently transfected with fulllength or 5=-deletion Pgp promoter constructs or cotransfected with Pgp promoter construct (pϪ1073/ϩ703) along with c-Fos and/or c-Jun expression vectors (13,48,49) by electroporation utilizing Amaxa technology and plated at a density of 1 ϫ 10 5 cells/cm 2 on 12-well collagen-coated plates (plastic supports). At 24 h posttransfection, cells were treated from the apical side with LA CS diluted in a ratio of 1:10 in cell culture medium supplemented with 1% FBS for 24 h. In experiments involving Erk1/2 MAPK phosphorylation, gel shift [electrophoretic mobility shift assay (EMSA)], and chromatin immunoprecipitation (ChIP) assays, Caco-2 cells plated at a density of 4 ϫ 10 4 cells/cm 2 on six-well plastic supports for 21 days were treated from the apical side with LA CS diluted in a ratio of 1:10 in cell culture medium supplemented with 1% FBS for 1, 3, or 24 h.…”

“…Caco-2 cells grown to confluence in six-well plastic supports (Corning Costar, Tewksbury, MA) were serum-starved overnight and treated with LA CS diluted in a ratio of 1:10 in cell culture medium supplemented with 1% FBS for 1, 3, or 24 h. Cells were washed three times with ice-cold PBS and lysed as described previously (48,49 Transient transfection and luciferase assays. Caco-2 cells were transfected with full-length or 5=-deletion Pgp promoter constructs and p-cytomegalovirus (CMV) ␤-galactosidase (pCMV␤) mammalian expression vector (BD Biosciences Clontech) by electroporation using the Amaxa Nucleofector system as described previously (48,49). Briefly, ϳ1 ϫ 10 6 cells were harvested and then electroporated in 100 l of Nucleofector Solution T (supplied by Amaxa) with one of the Pgp promoter-luciferase constructs (30 g) and 2.0 g of pCMV␤.…”

“…Transfected Caco-2 cells were treated from the apical side with LA CS diluted in a ratio of 1:10 in cell culture medium supplemented with 1% FBS for 24 h. At 48 h posttransfection, cells were washed with PBS and lysed using a passive lysis buffer (Promega). Pgp promoter activity was expressed in terms of relative luciferase activity normalized to ␤-galactosidase activity as described previously (48,49). In a separate set of experiments, Caco-2 cells were transiently cotransfected with 10 g of Pgp promoter construct (pϪ1073/ϩ703) or 20 g of c-Fos or c-Jun expression vector or a combination of c-Fos and c-Jun vectors along with 2.0 g of pCMV␤ per 24-well plate as described previously (13).…”

In &lt;i&gt;Vitro&lt;/i&gt; Effects of the Phytocomplex TrichoTech&lt;sup&gt;TM&lt;/sup&gt; on Human Fibroblasts: Proliferative Potential and Effects on Gene Expression of FGF-7 and FGF-10

“…Besides, no FGF-7 RNA was detected in follicles during catagen or telogen [23]. FGF-10 is found in the dermal papilla fibroblasts and its receptor FGFR2IIIb is found in the neighboring outer root sheath of the keratinocytes [24], suggesting that FGF-10 is a mesenchymally derived stimulator of hair follicle cells, which contribute to the hair-promoting activity. To verify the relative expression of mRNA for fibroblast growth factor-7 (FGF-7) and fibroblast growth factor-10 (FGF-10) genes, a real-time polymerase chain reaction (qPCR) protocol was used.…”