Lisa Choy

TGF‐β‐induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcin expression and inhibits osteoblast differentiation

Transforming growth factor‐β (TGF‐β), a secreted factor present at high levels in bone, inhibits osteoblast differentiation in culture; yet, the mechanism of this inhibition remains unclear. We studied the effects of TGF‐β and its effectors, the Smads, on the expression and function of the osteoblast transcription factor CBFA1. TGF‐β inhibited the expression of the cbfa1 and osteocalcin genes, whose expression is controlled by CBFA1 in osteoblast‐like cell lines. This inhibition was mediated by Smad3, which interacts physically with CBFA1 and represses its transcriptional activity at the CBFA1‐binding OSE2 promoter sequence. The repression of CBFA1 function by Smad3 contrasts with previous observations that Smads function as transcription activators. This repression occurred in mesenchymal but not epithelial cells, and depended on the promoter sequence. Smad3‐mediated repression of CBFA1 provides a central regulatory mechanism for the inhibition of osteoblast differentiation by TGF‐β, since it inhibits both cbfa1 transcription and transcriptional activation of osteoblast differentiation genes by CBFA1. Altering Smad3 signaling influenced osteoblast differentiation in the presence or absence of TGF‐β, implicating Smad3/TGF‐β‐mediated repression in autocrine regulation of osteoblast differentiation.

Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.

TGFβ‐stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro‐migratory TGFβ switch

During the course of breast cancer progression, normally dormant tumour‐promoting effects of transforming growth factor β (TGFβ), including migration, invasion, and metastasis are unmasked. In an effort to identify mechanisms that regulate the pro‐migratory TGFβ ‘switch’ in mammary epithelial cells in vitro, we found that TGFβ stimulates the phosphorylation of Smad1 and Smad5, which are typically associated with bone morphogenetic protein signalling. Mechanistically, this phosphorylation event requires the kinase activity and, unexpectedly, the L45 loop motif of the type I TGFβ receptor, ALK5, as evidenced by studies using short hairpin RNA‐resistant ALK5 mutants in ALK5‐depleted cells and in vitro kinase assays. Functionally, Smad1/5 co‐depletion studies demonstrate that this phosphorylation event is essential to the initiation and promotion of TGFβ‐stimulated migration. Moreover, this phosphorylation event is preferentially detected in permissive environments such as those created by tumorigenic cells or oncogene activation. Taken together, our data provide evidence that TGFβ‐stimulated Smad1/5 phosphorylation, which occurs through a non‐canonical mechanism that challenges the notion of selective Smad phosphorylation by ALK5, mediates the pro‐migratory TGFβ switch in mammary epithelial cells.

The type II transforming growth factor (TGF)-beta receptor-interacting protein TRIP-1 acts as a modulator of the TGF-beta response.

The transforming growth factor-β (TGF-β) receptor interacting protein TRIP-1 was originally identified as a WD40 repeat-containing protein that has the ability to associate with the TGF-β type II receptor and is phosphorylated by it (1). However, its function was not known. We now show that TRIP-1 expression represses the ability of TGF-β to induce transcription from the plasminogen activator inhibitor-1 promoter, a common reporter of the TGF-β-induced gene expression response, but does not affect the ability of TGF-β to inhibit cyclin A transcription. TRIP-1 can also inhibit the plasminogen activator inhibitor-1 expression induced by Smads as well as activated TGF-β type I receptors. Its inhibitory effect is exerted by a combination of receptor-dependent and receptor-independent mechanisms. Deletion mutational analysis revealed that two distinct regions, which do not contain recognizable WD40 repeats, are required for the ability of TRIP-1 to inhibit the gene expression response. Expression of other segments of TRIP-1 increased the TGF-β-induced gene expression response and therefore may exert a dominant negative phenotype. We conclude that TRIP-1 acts as a modulator of the TGF-β response.

Transforming Growth Factor β/Smad3 Signaling Regulates IRF-7 Function and Transcriptional Activation of the Beta Interferon Promoter

ABSTRACT The rapid induction of alpha interferon (IFN-α) and IFN-β expression plays a critical role in the innate immune response against viral infection. We studied the effects of transforming growth factor β (TGF-β) and its intracellular effectors, the Smads, on the function of IRF-7, an essential transcription factor for IFN-α and -β induction. IRF-7 interacted with Smads, and IRF-7, but not IRF-3, cooperated with Smad3 to activate IFN-β transcription. This transcriptional cooperation occurred at the IRF-binding sequences in the IFN-β promoter, and dominant-negative interference with TGF-β receptor signaling and Smad3 function decreased IRF-7-mediated transcription. Furthermore, elimination of Smad3 expression in Smad3−/− fibroblasts delayed and decreased double-stranded RNA-induced expression of endogenous IFN-β, whereas restoration of Smad3 expression enhanced IFN-β induction. The IRF-7-Smad3 cooperativity resulted from the regulation of the transactivation activity of IRF-7 by Smad3, and dominant-negative interference with Smad3 function decreased IRF-7 activity. Consistent with the regulation by Smad3, the transcriptional activity of IRF-7 depended on and was regulated by TGF-β signaling. Our studies underscore a role of TGF-β/Smad3 signaling in IRF-7-mediated induction of IFN-β expression.

21 TGF-β Family Signaling in Mesenchymal Differentiation

Signaling by transforming growth factor-β (TGF-β) family factors affects many distinct differentiation pathways, including those of the hematopoietic and immune cell lineages, epithelial lineages, and hemangioblasts. Similarly, TGF-β family signaling has revealed that it is a major regulatory network that drives lineage selection and differentiation progression in mesenchymal cells. As with other systems, the signaling activity of TGF-β family members correlates with the temporal and spatial regulation of TGF-β family ligand, receptor, and Smad expression, each of which is regulated during mesenchymal cell differentiation. Likewise, the activities of the ligands, receptors, and Smads are also regulated during differentiation. For example, ligand activity is dictated by secreted antagonists or ligand-binding proteins. Receptors are activated through autocrine and paracrine signaling. Smad activity is defined by combinatorial interactions and cross-talk with other signaling pathways, and the Smads then serve to integrate information from a multitude of signaling pathways that direct the expression and activity of each component of the TGF-β signaling pathway. In this way, the TGF-β family receptors and Smads act as cell-intrinsic regulators of mesenchymal differentiation. In this chapter, we discuss the roles of TGF-β family signaling in mesenchymal differentiation. MESENCHYMAL DIFFERENTIATION The mesenchyme consists of loosely associated stellate-shaped cells, which in the trunk and posterior regions of the head derive from mesoderm, and in the face, jaws, and neck, originate from the neural crest (Noden 1986; Couly et al. 1992; Olivera-Martinez et al. 2000; Matsuoka et al. 2005). Neural crest mesenchyme arises from the dorsal edges of the neuroepithelium during...

Smads In Mesenchymal Differentiation

Transforming growth factor-β (TGF-β) family members play key roles in development through their regulatory roles in cell and tissue differentiation. Among the differentiation lineages, mesenchymal tissue differentiation into bone, fat, cartilage or muscle is strongly regulated by TGF-β family members. Smads have shown themselves to function as cell-intrinsic regulators of mesenchymal stem cell differentiation into osteoblasts, adipocytes, chondrocytes and myocytes. Their activities are defined by autocrine and paracrine signals from TGF-β family members, and regulate the proliferation of the mesenchymal progenitor cell pool, the selection of the lineage along which the cells will differentiate, and the progression of differentiation. At the molecular level, Smads can inhibit progression of differentiation, through functional repression of key transcription factors that drive differentiation, or alternatively activate the expression, or enhance the activities, of such transcription factors to drive the selection of a lineage and progression along a particular lineage