A Truncated Serpin D1 Variant Regulates Adseverin-Dependent Actin Severing and Is Required for Osteoclastogenesis.

Adseverin is a Ca2+-dependent, actin severing protein that promotes fusion of osteoclast precursors in osteoclast formation. Currently it is not understood how actin severing activity is regulated in osteoclastogenesis. Mass spectrometry of adseverin immunoprecipitates from osteoclasts showed that adseverin associated with Serpin D1. Purified Serpin D1 interacted with adseverin in vitro in a Ca2+-dependent manner. Ca2+ increased actin severing by ~2-fold. Cells that differentiated into osteoclasts expressed an intracellular, truncated Serpind1 isoform that lacked exons 1 and 2, and part of exon 3. This truncated Serpin D1 isoform was distinct from the secreted, full-length Serpin D1 protein that inhibits thrombin activity. The expression of truncated Serpin D1 in RAW264.7 cells appeared to be further increased following TNF-α treatment during RANKL-induced osteoclastogenesis. CRISPR/Cas9-mediated partial deletion of exon 3 of Serpind1, which resulted in Serpind1 knockout, increased the abundance of subcortical actin filaments. This treatment also altered the spatial distribution of adseverin, inhibited the expression of osteoclast-specific genes and reduced the formation of multinucleated osteoclasts by >90% in vitro. We conclude that a truncated, intracellular variant of Serpin D1 interacts with adseverin to promote actin severing and osteoclast formation.