Enhancing collagen biosynthesis in mammalian cells through hypoxia-mimetic prolyl hydroxylase inhibition

Abstract Collagen, the most abundant protein in the extracellular matrix of mammalian cells, is a key biomaterial in various biotechnological and therapeutic applications, such as tissue engineering and regeneration, cosmetics, and cultivated meat. Despite the increasing demand for culture-based collagen, it is still mainly produced from animal connective tissues. A recent study has shown that under hypoxia, activation of the hypoxia-inducible factor (HIF) leads to enhanced type I collagen biosynthesis. However, under normal oxygen conditions, HIF activity is downregulated by the HIF-prolyl hydroxylase (PHD) enzyme. Based on these previous findings, we hypothesized that inhibiting PHD activity by exogenous modulators could mimic hypoxia, leading to elevated HIF transcriptional activity and enhancing collagen biosynthesis under normoxia. Our study demonstrates that pharmacological PHD inhibition using a set of small molecules targeting their 2-oxoglutarate (2-OG)-dependent activity is associated with enhanced HIF activity, upregulation of key-collagen modifying enzymes, including collagen prolyl 4-hydroxylases and lysyl hydroxylases, accompanied by a 10- to 29-fold increase in collagen type I fluorescence intensity in NIH/3T3 embryonic mouse fibroblast cells. These findings show that targeting PHD can effectively enhance collagen production in mammalian cells, suggesting that the modulation of intracellular key protein signaling pathways presents a promising strategy for enhancing the production of culture-based collagen.