A Low-Phenylalanine-Containing Whey Protein Hydrolysate Stimulates Osteogenic Activity through the Activation of p38/Runx2 Signaling in Osteoblast Cells

A phenylalanine (Phe)-restricted diet is indispensable for individuals suffering from phenylketonuria (PKU). Our previous study reported a low-Phe-containing whey protein hydrolysate (LPH) prepared from a selected whey protein hydrolysate (TA2H). This study aimed to investigate the osteogenic activity of LPH and TA2H in MC3T3-E1 preosteoblast cells and explore the underlying mechanism. Results showed that the treatment of TA2H and LPH (at the final concentrations of 100–1000 μg/mL) had a stimulatory effect on the proliferation, differentiation, and mineralization of MC3T3-E1 cells. The LPH of 1000 μg/mL significantly increased cell proliferation (2.15- ± 0.11-fold) and alkaline phosphatase activity (1.22- ± 0.07-fold), promoted the protein and mRNA levels of runt-related transcription factor 2 (Runx2, 2.50- ± 0.14-fold and 2.97- ± 0.23-fold, respectively), enhanced the expression of differentiation biomarkers (type-I collagen, osteocalcin, and osteopontin), increased calcium deposition (1.56- ± 0.08-fold), and upregulated the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand. The exploration of signaling pathways indicated that the activated p38-dependent Runx2 signaling contributed to the LPH-induced osteogenesis. These results provided evidence, for the first time, that a prepared low-Phe whey protein hydrolysate positively modulated the activity of osteoblasts through the p38/Runx2 pathway, thereby providing a new osteoinductive protein substitute to make functional PKU food.