Micellar Casein and Whey Powder Hold a TGF-β Activity and Regulate ID Genes In Vitro

Abstract

Casein and whey being food supplements have been considered to be used in oral health care products. However, the response of oral cells to micellar casein and whey powder remains unclear. Considering that milk contains the growth factor TGF-β, and lactoperoxidase was recently reported to decrease the expression of inhibitor of DNA-binding (ID) proteins, there is a rationale to assume that casein and whey can also provoke these responses in oral cells. To examine the TGF-β activity, gingival fibroblasts were exposed to reconstituted casein and whey powder from food supplement before the expression of TGF-β target genes were analyzed by reverse transcription-quantitative polymerase chain reaction. Immunoassays were performed for interleukin11 (IL11) in the cell culture supernatant and for TGF-β in the reconstituted casein and whey. We blocked TGF-β by neutralizing the antibody and the TGF-β receptor type I kinase with the inhibitor SB431542. We also showed smad3 phosphorylation and smad2/3 nuclear translocation by Western blot and immunostaining, respectively. Moreover, with reconstituted casein and whey powder, ID1 and ID3 expression analysis was evaluated in HSC2 human oral squamous carcinoma cells. We report here that casein and whey powder caused a robust increase of TGF-β target genes interleukin11 (IL11), NADPH oxidase 4 (NOX4) and proteoglycan4 (PRG4) in gingival fibroblasts that was blocked by SB431542 and the neutralizing antibody. Moreover, casein and whey powder increased the phosphorylation of smad3 and nuclear translocation of smad2/3. No changes of proliferation markers Ki67 and cyclinD1 were observed. Furthermore, reconstituted casein and whey powder decreased ID1 and ID3 expression in the HSC2 oral squamous carcinoma cells. These findings suggest that the processing of milk into casein and whey powder maintains the TGF-β activity and its capacity to regulate ID1 and ID3 genes in oral fibroblasts and oral squamous carcinoma cells, respectively. These data increase the scientific knowledge on the biological activity of casein and whey with a special emphasis on oral health.

Keywords: TGF-β; casein; epithelial cells; fibroblasts; nutrition; oral health; whey.

Figure 1

Casein and whey powder at 1% do not affect the viability of gingival fibroblasts. Gingival fibroblasts were incubated for 24 h with 0.1% to 10% (A) casein and (B) whey. Substrate conversion into solubilized formazan crystals was determined on a photometer. Graphs showing the formation of formazan crystals being expressed as percentage of unstimulated controls (100%). Statistic was based on a Friedmann test.

Figure 2

Casein and whey powder at 1% have no impact on cell proliferation. Gingival fibroblasts were incubated for 24 h with 1% casein and whey powder, as well as with 100 ng/mL of the mitogens PDGF-BB and 50 ng/mL bFGF. (A) Immunostaining for Ki-67 protein (also known as MKI67), a cellular marker for proliferation and (B) the expression of the cell cycle regulator cyclin D1 (CCND1) were determined. Data were normalized for the untreated control being “one”. Statistic was based on a Friedmann test. Scale bar indicates 100 µm. “wo” stands for without, indicating the unstimulated cells.

Figure 3

Casein and whey powder enhance TGF-β target genes in gingival fibroblasts. Gingival fibroblasts were exposed to 1% of a pooled reconstituted powder from three providers of casein and whey for 24 h followed by expression analysis of (A) IL11, (B) NOX4 and (C) PRG4. Data points represent fold change of independent experiments compared to the unstimulated controls. Statistical analysis was based on Mann–Whitney U test.

Figure 4

TGF-β pan specific neutralizing antibody reduces whey TGF-β activity. Gingival fibroblasts were exposed to 1% whey powder with and without 10 ng/mL of a TGF-β pan specific neutralizing antibody for 24 h followed by expression analysis of (A) IL11, (B) NOX4 and (C) PRG4. Data points represent fold change of independent experiments compared to the unstimulated controls. Statistical analysis was based on paired T-test.

Figure 5

Casein and whey powder formulation enhances TGF-β target genes via TGF-β RI kinase. Gingival fibroblasts were exposed to 1% of a reconstituted pooled casein (A–C) and whey (D–F) powder with and without the TGF-β RI kinase inhibitor SB431542 (SB43). Expression analysis of (A,D) IL11, (B,E) NOX4 and (C,F) PRG4 was performed with RT-PCR. Data points represent fold change of independent experiments compared to the unstimulated controls. Statistical analysis was based on paired T-test.

Figure 6

IL11 immunoassay of fibroblasts exposed to cell supernatants. Immunoassay for IL11 was performed with the supernatant of the cells and the data are expressed as pg/mL. TGF-β RI kinase inhibitor SB431542 is marked as SB43. Statistical analysis was based on a Friedman test and Dunn’s multiple comparison. “wo” stands for without, indicating the unstimulated cells.

Figure 7

Casein and whey powder enhance phosphorylation of smad3. Serum-starved gingival fibroblasts were exposed to 1% of a reconstituted pooled casein and whey powder for 30 min before being subjected to Western blot analysis of phosphorylation of smad3. Cells exposed to recombinant TGF-β and casein and whey powder caused a strong increase in the phosphorylation of smad3. “wo” stands for without, indicating the unstimulated cells.

Figure 8

Casein and whey powder enhance smad2/3 nuclear translocation. Serum-starved gingival fibroblasts were exposed to TGF-β (10 ng/mL) and 1% of a casein and whey powder for 30 min before fluorescent labelling of smad2/3. The nuclear signal is visible with cells exposed to recombinant TGF-β and casein and whey powder. Scale bar indicates 100 µm. “wo” stands for without, indicating the unstimulated cells.

Figure 9

ID1 and ID3 expression in HSC2 exposed to casein and whey powder. HSC2 oral squamous cell carcinoma cells were exposed to 1% casein and whey powder for 24 h, before expression analysis of the target genes (A) ID1 and (B) ID3 were performed. Data indicate the x-fold decrease compared to unstimulated control cells. Statistical analysis was based on Mann–Whitney U test.