Abstract
Collagen-derived hydroxyproline (Hyp)-containing peptides have a variety of biological effects on cells. These bioactive collagen peptides are locally generated by the degradation of endogenous collagen in response to injury. However, no comprehensive study has yet explored the functional links between Hyp-containing peptides and cellular behavior. Here, we show that the dipeptide prolyl-4-hydroxyproline (Pro-Hyp) exhibits pronounced effects on mouse tendon cells. Pro-Hyp promotes differentiation/maturation of tendon cells with modulation of lineage-specific factors and induces significant chemotactic activity in vitro. In addition, Pro-Hyp has profound effects on cell proliferation, with significantly upregulated extracellular signal–regulated kinase phosphorylation and extracellular matrix production and increased type I collagen network organization. Using proteomics, we have predicted molecular transport, cellular assembly and organization, and cellular movement as potential linked-network pathways that could be altered in response to Pro-Hyp. Mechanistically, cells treated with Pro-Hyp demonstrate increased directional persistence and significantly increased directed motility and migration velocity. They are accompanied by elongated lamellipodial protrusions with increased levels of active β1-integrin–containing focal contacts, as well as reorganization of thicker peripheral F-actin fibrils. Pro-Hyp–mediated chemotactic activity is significantly reduced (p < 0.001) in cells treated with the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or the α5β1-integrin antagonist ATN-161. Furthermore, ATN-161 significantly inhibits uptake of Pro-Hyp into adult tenocytes. Thus, our findings document the molecular basis of the functional benefits of the Pro-Hyp dipeptide in cellular behavior. These dynamic properties of collagen-derived Pro-Hyp dipeptide could lead the way to its application in translational medicine.
Highlights
Collagen is the most abundant extracellular matrix (ECM) protein in tissue/organ stroma and significantly contributes to tissue/organ integrity [1]
Pro-Hyp showed the greatest activity in adult tenocytes and tendon progenitor cells at day 3, which was accompanied by significant upregulation of type I collagen and fibronectin mRNA levels (Fig. S1, A and B). mRNA of the tendon-specific transcription factor Scx was significantly upregulated at day 6 (Fig. S1B)
We found that higher concentrations, 200 and 500 μg/ml, showed more effective cell proliferation activity in both tenocytes and tendon progenitor cells (Fig. 1A)
Summary
Collagen is the most abundant extracellular matrix (ECM) protein in tissue/organ stroma and significantly contributes to tissue/organ integrity [1]. Pro-Hyp showed the greatest activity in adult tenocytes and tendon progenitor cells at day 3, which was accompanied by significant upregulation of type I collagen and fibronectin mRNA levels (Fig. S1, A and B).
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