Transforming Growth Factors β Coordinate Cartilage and Tendon Differentiation in the Developing Limb Mesenchyme

Abstract

Transforming growth factor ␤ (TGF␤) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGF␤ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGF␤s shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved downregulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGF␤ signaling up-regulates Sox9 in the in vivo experimental model system in which TGF␤ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGF␤ signaling, we found that TGF␤s appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGF␤ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGF␤ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGF␤ treatments. TGF␤s4 form a small family of multipotent regulatory polypeptides that gives the name to a large cytokine superfamily, which also includes Activins and bone morphogenetic proteins, characterized by structural and signaling similarities. In mammalians and birds the family is composed of three highly homologous isoforms, although the avian homologue of the mammalian TGF␤1 was initially named TGF␤4 (1). Signaling by these factors is mediated by ligand binding with type II and type I receptors to form heteromeric complexes, which phosphorylate Smad2 and -3 proteins. The activated p-Smads associate with Smad4 prior to translocation into the nucleus, where they regulate the expression of a variety of genes (see Ref. 2). TGF␤s may also signal through the mitogen-activated protein kinase (MAPK) transduction pathway (3).TGF␤s are considered major regulators of the differentiation and growth of the skeletal connective tissues, with a promising future in regenerative medicine as tools to modulate the differentiation of stem cells for reconstruction of cartilage, tendon, or bone. In the long appendicular bones TGF␤s are expressed in the chondrocytes of the growth plate and regulate the rate of differentiation of prehypertrophic chondrocytes to hypertrophic chondrocytes (4 -7). In embryonic systems, TGF␤s are expressed in the developing limb forming well defined domains in the digit chondrogenic aggregates, in the developing joints and in the differentiating tendon blastema...

Anterior cruciate ligament-derived cells have high chondrogenic potential

“…The BMP inhibitor Noggin represses the expression of Sox9 while inducing the Scx expression [35]. In micromass cultures of undifferentiated mesodermal cells, TGF-β/Smad signaling is a direct inducer of Sox9 and Scx, but transcriptional repressors of TGF-β signaling (Tgif1 and SnoN) modulate the expression of Sox9 and Scx [36]. These findings suggest that TGF-β superfamily modulate the balance between Sox9 and Scx according to each differentiation stage of chondrogenesis.…”

Enrichment of Human ESC-Derived Multipotent Mesenchymal Stem Cells with Immunosuppressive and Anti-Inflammatory Properties Capable to Protect Against Experimental Inflammatory Bowel Disease

“…The transforming growth factor (TGF-b) signaling through SMAD-2/3 downstream effectors has an increasing interest in regenerative medicine and lineage specification during human embryonic development [15,16]. In contrast to specific bone morphogenic proteins (BMPs), which are known to play an important role in directing cell fate decisions toward mesoderm and further differentiation into MSCs, the mesodermal effects of TGF-b signaling in human embryonic development is controversial [15,16].…”

Three-Dimensional Culture and Transforming Growth Factor Beta3 Synergistically Promote Tenogenic Differentiation of Equine Embryo-Derived Stem Cells

“…4 Multiple studies have shown that TGF-b is up-regulated after tendon injury [5][6][7] and after their injection into the injured tendon, pluripotent equine embryo-derived stem cells (ESCs) express SCX. 5 The addition of TGF-b3 induces tenogenic differentiation of in vitro cultures of mesenchymal stem cells (MSCs), 8 micromass cultures of undifferentiated limb mesenchyme, 9 and equine ESCs. 5 Taken together, this suggests that TGF-b signaling plays a key role in triggering the initiation of tenocyte differentiation from stem cells and tendon progenitors.…”