Antagonism of BMP signaling is insufficient to induce fibrous differentiation in primary sclerotome

Abstract

Sclerotome is the embryonic progenitor of the axial skeleton. It was previously shown that Tgfbr2 is required in sclerotome for differentiation of fibrous skeletal tissues including the annulus fibrosus of the intervertebral disc. Alternatively, BMP signaling is required to form the vertebral body through chondrogenesis. In addition, TGFβ added to sclerotome cultures induces expression of markers for fibrous tissue differentiation but not cartilage or bone. The mechanism of how TGFβ signaling regulates this lineage decision in sclerotome is not known and could be due to the production of instructive or inhibitory signals or a combination of the two. Here we show that TGFβ antagonizes BMP/ Smad1/5 signaling in primary sclerotome likely through regulation of Noggin, an extracellular BMP antagonist, to prevent chondrogenesis. We then tested whether inhibition of BMP signaling, and inhibition of chondrogenesis, is sufficient to push cells toward the fibrous cell fate. While Noggin inhibited BMP/ Smad1/5 signaling and the formation of chondrogenic nodules in sclerotome cultures; Noggin and inhibition of BMP signaling through Gremlin or DMH2 were insufficient to induce fibrous tissue differentiation. The results suggest inhibition of BMP signaling is not sufficient to stimulate fibrous tissue differentiation and additional signals are likely required. We propose that TGFβ has a dual role in regulating sclerotome fate. First, it inhibits BMP signaling potentially through Noggin to prevent chondrogenesis and, second, it provides an unknown instructive signal to promote fibrous tissue differentiation in sclerotome. The results have implications for the design of stem cell-based therapies for skeletal diseases.

Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

“…To determine if Scx expression was required to regulate Fmod and Adamtsl2, we inhibited Scx synthesis by transducing C 3 H 10 T 1/2 cells, a murine mesenchymal stem cell line, with 30 pM of Scx specific siRNA or a 30 pM of Scrambled (Scram) siRNA as a control. We and others have previously shown that fibrous genes are regulated by TGFβ1 in C 3 H 10 T 1/2 cells 32,33 . Western blot analysis showed a significant decrease in Scx protein expression in Scx siRNA transduced cells when compared to Scram control (Fig.…”

“…We previously identified several TGF-β regulated genes for which expression is enriched in the putative IVD region of the embryonic sclerotome, including Adamtsl2, Fmod, and Scx 18 , 31 , 34 . Localization to a patterned subset of sclerotome was previously confirmed by in-situ hybridization 18 , 35 .…”

Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

“…To determine if Scx expression was required to regulate Fmod and Adamtsl2, we inhibited Scx synthesis by transducing C 3 H 10 T 1/2 cells, a murine mesenchymal stem cell line, with 30pM of Scx specific siRNA or a 30pM of Scrambled (Scram) siRNA as a control. We and others have previously shown that fibrous genes are regulated by TGFβ1 in C 3 H 10 T 1/2 cells 32,33 . Western blot analysis showed a significant decrease in Scx protein expression in Scx siRNA transduced cells when compared to Scram control (Fig.…”

“…We previously identified several TGF-b regulated genes for which expression is enriched in the putative IVD region of the embryonic sclerotome, including Adamtsl2, Fmod, and Scx 18,31,34 . Localization to a patterned subset of sclerotome was previously confirmed by in-situ hybridization 18,35 .…”

“…More recently, it was shown that loss of Scx disrupts maturation of AF, and the loss of Sox9 in Scx expressing cells disrupts formation of the AF and chondro-tendinous/ligamentous junctions 38,39 . Scx expression is up-regulated by TGF-β in mesenchymal cells including primary sclerotome and C 3 H 10 T 1/2 cells and down-regulated in tissues in which Tgfbr2 has been conditionally deleted 8,31,34 . bHLH transcription factors like Scx generally bind to DNA as dimers to elements known as E-boxes 40 .…”