# Protein Kinase R-like Endoplasmic Reticulum Kinase-Mediated ER-Mitochondria Coupling Regulates Odontogenic Differentiation of Human Dental Pulp Stem Cells Under Inflammatory Stimuli

## TL;DR

This study shows how PERK in MAMs regulates the differentiation of dental pulp stem cells under inflammation, affecting calcium signaling and cell function.

## Contribution

The study reveals a novel PERK-centered mechanism in MAMs that controls inflammation-impaired odontogenic differentiation of hDPSCs.

## Key findings

- LPS activates PERK, increasing MAM-related molecules and ER-mitochondria coupling.
- PERK silencing reduces mitochondrial and ER damage and restores odontogenic potential.
- PERK suppresses mineralization by modulating IP3R-mediated calcium signaling in MAMs.

## Abstract

Human dental pulp stem cells (hDPSCs) play pivotal roles in the regeneration of pulp-dentin complex, yet their odontogenic differentiation is critically modulated by the inflammatory microenvironment. Protein kinase R-like endoplasmic reticulum kinase (PERK), a key regulator of endoplasmic reticulum stress, is highly enriched in mitochondria-associated endoplasmic reticulum membranes (MAMs) and exerts critical functions. However, its precise mechanisms in inflammatory regulation and cellular differentiation remain elusive. This study elucidates the PERK-centred regulatory mechanism in MAMs that governs inflammation-impaired odontogenic differentiation of hDPSCs, potentially involving IP3R-dependent calcium flux and dynamic protein interactions in MAMs.

Rat pulpitis models and in vitro lipopolysaccharide (LPS)-induced inflammatory models of hDPSCs were established to investigate the effects of PERK signalling in odontogenesis under inflammatory conditions. Lentivirus-mediated silencing of PERK was performed to evaluate its role in LPS-induced inflammation. Molecular mechanisms were analysed using RNA sequencing, immunofluorescence, and transmission electron microscopy analyses.

LPS stimulation activated the PERK signalling pathway, significantly upregulating MAM-related molecules (IP3R, VDAC1, GRP75) and enhancing PERK/VDAC1 colocalization and the formation of endoplasmic reticulum-mitochondria coupling structures. PERK silencing effectively mitigated LPS-induced mitochondrial swelling, ER dilatation, and calcium influx dysregulation, while restoring alkaline phosphatase activity and odontogenic differentiation potential. Mechanistically, PERK suppressed hDPSC mineralization by modulating IP3R-mediated calcium signalling pathway in MAMs.

This study demonstrates that LPS-induced inflammatory stress reprograms hDPSCs bioactivity via PERK-centric control of MAMs likely through quantitative enhancement, structure specialization, and functional potentiation. The underlying mechanisms may involve IP3R-mediated regulation of calcium ion influx and protein interactions within MAMs.