To investigate the role of klotho shedding in the ageing process

Ageing is the most important risk factor for the majority of human pathologies. Klotho is one of the well-known anti-ageing proteins that exists in two major forms including the full-length membrane form (mKl) and a shed soluble form (sKl). The biological functions and mechanism of action of mKl have been well-explored. By contrast, the function of sKl and the mechanism regulating its secretion are less clear.

To explore the mechanism underlying the proteolytic shedding of klotho, we put our focus on Membrane-type 1 Matrix Metalloproteinase (MT1-MMP/MMP14), a membrane-bound protease responsible for extracellular matrix remodeling and pericellular proteolysis. Mmp14-/- mice manifest a variety of ageing-like phenotypes resembling those observed in klotho deficient mice and human ageing. Despite a striking similarity in ageing-like phenotypes between Mmp14-/- and kl-/- mice, the exact role of MT1-MMP in ageing process and whether MT1-MMP regulates the activity of klotho remain unknown. Our preliminary findings along with other studies showed that the loss of MT1-MMP induces senescent phenotypes, including increased activity of senescence‐associated β‐galactosidase and upregulation of p16INK4a and p21CIP1/WAF1, two biomarkers of cellular senescence. We further found a significant reduction in the level of serum α-klotho in Mmp14-/- mice. Consistently, the soluble form of α-klotho was greatly reduced in the conditioned medium derived from Mmp14-/- kidney slices cultured ex vivo whereas the expression of total α-klotho in kidney homogenates was slightly increased by the loss of MT1-MMP, suggesting that MT1- MMP may regulate klotho shedding. The regulation of klotho by MT1-MMP was further substantiated by their physical interaction. Based on these new findings, we hypothesize that MT1-MMP may regulate the bioavailability of soluble klotho by ectodomain shedding, thereby suppressing systematic cellular senescence and ageing.

To further explore the interplay between MT1-MMP and klotho, we propose to investigate (1) the molecular mechanism by which MT1-MMP sheds klotho; (2) whether supplementation of klotho ameliorates the ageing-like phenotypes of Mmp14-/- mice and extends their lifespan; (3) whether blockage of klotho cleavage reduces the bioavailability of soluble klotho and leads to premature ageing. The information derived from this study not only provide new insights into the biological functions of skl, but will also identify the potential mechanism boosting the secretion of skl. This could lay a foundation for innovative approaches to promoting healthy ageing and increasing longevity