The biological relevance of Klotho in aging processes was impressively demonstrated in transgene and knock out mouse experiments and its impact on calcium, vitamin D and phosphate metabolism suggests multifunctional modes of action.
The human alpha Klotho gene encodes two transcripts, one for a protein of 130 kDa with a short intracellular and a transmembrane region and the two extracellular domains KL1 and KL2. A second transcript exists due to alternative splicing, encoding a secreted protein of 70 kDa with sequence identity to KL1, but differing in the last 15 amino acid residues. Beyond that, the transmembrane region can be cleaved by metalloproteinases, generating a circulating Klotho protein whose domains KL1 and KL2 can furthermore be proteolytically separated. This does not only multiply the modes of action of Klotho, the high sequence identity makes it also difficult to analyze differences between the two transcript variants. Specific antibodies, which are able to distinguish between the KL1 domains, generated by proteolytic cleavage or by alternative splicing, are necessarily required. As yet it is unknown if specific receptors exist to transduce biologic effects of the KL1 domains.
We recently established a stable cell line expressing the alternatively spliced KL1 domain and analyzed its effect on human mesenchymal stem cells (hMSC), alone and in combination with FGF23. Furthermore, we characterized a new antibody generated by Immundiagnostik AG, which is specific for this Klotho variant. This enabled us to analyze endogenous expression and intracellular localization of Klotho in hMSC.
Immunocytochemical analyses of HEK293 cells transfected with expression plasmids coding for membrane bound or secreted Klotho, respectively, clearly demonstrated the specificity of the new antibody. We could detect endogenous expression of this Klotho variant in hMSC by RT-PCR and immunocytochemistry, however donor-dependent differences were observed. Stimulation of hMSC with secreted Klotho can induce expression of egr1, not only in combination with FGF23, even admitted alone. Furthermore, stimulation with labeled secreted Klotho suggests an uptake of extracellular Klotho into the cell, but the mechanism remains unknown.
These observations presume an autocrine/paracrine mechanism in hMSC, possibly with impact on the wnt-pathway, since slight induction of connexin 43 was measured in qPCR.
14 - 17 May 2016
European Calcified Tissue Society