# GNAS/PKA signaling promotes aberrant osteochondral differentiation of Gli1+ tendon sheath progenitors

## TL;DR

This study identifies a specific group of tendon stem cells that can turn into bone and cartilage after injury, and shows that a signaling pathway called GNAS/PKA is key to this process.

## Contribution

The novel contribution is the discovery that Gli1+ tendon sheath progenitors can undergo osteochondral differentiation via GNAS/PKA signaling, offering a new therapeutic target for tendon ossification.

## Key findings

- Gli1+ tendon sheath cells expand and differentiate into osteochondral lineages after injury.
- GNAS/PKA signaling is activated during this differentiation process.
- Inhibiting PKA or using NF449 reduces aberrant osteochondral differentiation in mice and human cells.

## Abstract

Tendon injury promotes aberrant osteochondral differentiation of tendon stem cells (TSCs) and results in disability. However, the cellular subsets within the osteochondral lineage involved in this process and associated mechanisms remain unclear. Here, we found that, following Achilles tenotomy, murine Gli1+ tendon sheath cells expanded rapidly, transitioning into tenogenic and osteochondrogenic cells. Lineage tracing, together with single-cell RNA sequencing, revealed that osteochondrogenic Gli1+ tendon sheath cells originate from Scx+ tendon stem/progenitor cells, preferentially differentiate into osteochondral lineage tendon progenitors at 7 dpi, subsequently undergoing aberrant chondrogenesis and osteogenesis at 21dpi and 63dpi, respectively. In addition, Acvr1R206H/+ robustly accelerates osteochondral differentiation in Gli1+ tendon sheath progenitors. Furthermore, GNAS/PKA signaling was significantly activated in osteochondral differentiation of Gli1+ tendon sheath progenitors. Alternatively, treatment with the Gsα antagonist, NF449, or genetic inhibition of the PKA subunit, Prkaca, in Gli1+ sheath progenitors significantly alleviated aberrant osteochondral differentiation. NF449 also prevented osteochondral differentiation of human tendon stem cells. These findings identify Gli1+ tendon sheath progenitors with osteochondral differentiation capacity during heterotopic ossification via activation of GNAS/PKA signaling, suggesting PKA as a potentially effective therapeutic target to treat tendon ossification.

Injury of tendons triggers aberrant osteochondral differentiation of mesenchymal cells, leading to aberrant bone formation. This study identifies Gli1+/Scx+ tendon sheath progenitors as an alternative source of injury-induced ossification in the murine tendon.

Gli1+ tendon sheath cells are a subpopulation of tendon stem/progenitor cells.Gli1+/Scx+ tendon sheath progenitors possess the capacity for osteochondral differentiation.Acvr1R206H/+ mutation accelerates osteochondral differentiation of Gli1+ tendon sheath progenitors.GNAS/PKA/CREB signaling determines the osteochondral differentiation of Gli1+ tendon sheath progenitors.

Gli1+ tendon sheath cells are a subpopulation of tendon stem/progenitor cells.

Gli1+/Scx+ tendon sheath progenitors possess the capacity for osteochondral differentiation.

Acvr1R206H/+ mutation accelerates osteochondral differentiation of Gli1+ tendon sheath progenitors.

GNAS/PKA/CREB signaling determines the osteochondral differentiation of Gli1+ tendon sheath progenitors.

Gli1+/Scx+ sheath progenitors contribute to injury-induced ossification in the murine tendon.