Tgfbr2 regulates the maintenance of boundaries in the axial skeleton.

Previously, we showed that deletion of the TGF-beta type II receptor (Tgfbr2) in Type II Collagen (Col2a) expressing cells results in defects in the development of the axial skeleton. Defects included a reduction in size and alterations in the shape of specific vertebral elements. Anterior lateral and dorsal elements of the vertebrae were missing or irregularly shaped. Vertebral bodies were only mildly affected, but the intervertebral disc (IVD) was reduced or missing. In this manuscript, we show that alterations in the initiation or proliferation of cartilage are not detected in the axial skeleton. However, the expression domain of Fibromodulin (Fmod), a marker of the IVD, was reduced and the area of the future IVD contained peanut agglutinin (PNA) staining cartilage. Next, we show that the expression domains of Pax1 and Pax9, which are preferentially expressed in the caudal sclerotome, are expanded over the entire rostral to caudal length of the sclerotome segment. Dorsal-ventral patterning was not affected in these mice as accessed by expression of Pax1, Pax9, and Msx1. Proliferation was modestly reduced in the loose cells of the sclerotome. The results suggest that signaling through Tgfbr2 regulates the maintenance of boundaries in the sclerotome and developing axial skeleton.

Transforming growth factor-beta signaling helps specify tumor type in DMBA and hormone-induced mammary cancers.

To determine the role of transforming growth factor-beta (TGF-beta) signaling in mammary development and tumor formation, we previously generated transgenic mice that expressed a dominant-negative form of the TGF-beta type II receptor (DNIIR) under the control of DNA regulatory elements from the metallothionein promoter (MT-DNIIR-28). In this report, we tested the hypothesis that loss of TGF-beta signaling in the mammary gland alters the development of chemically or hormonally induced tumors in mice. Four groups of mice were used in the study: wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft, and wild-type and MT-DNIIR-28 mice on zinc with pituitary isograft treated with the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). Tumor-free survival over time, tumor growth rate, and tumor pathology were measured. Statistically significant differences in tumor free survival over time or tumor growth rate were not detected in wild-type versus transgenic mice treated with DMBA. In contrast, tumor-free survival was significantly altered in transgenic mice that were treated with the pituitary isograft alone with MT-DNIIR mice developing tumors more quickly. Alterations in the types of tumors that formed in wild-type versus MT-DNIIR DMBA-treated mice were detected. In wild-type mice, tumors with squamous differentiation or bicellular adenomyoepitheliomas were most common. Adenomyoepitheliomas were not detected in transgenic mice. Furthermore, there was reduced staining for alpha smooth muscle actin and keratin 14, markers for myoepithelial cells, in the glandular portion of tumors in transgenic mice. The pathology of tumors induced by pituitary isograft alone was also markedly different in wild-type and transgenic mice. All the tumors classified from wild-type mice demonstrated some form of squamous differentiation, whereas squamous differentiation was not detected in the pituitary-induced transgenic tumors. The results suggest that TGF-beta acts as a tumor suppressor for hormone-induced cancers and that TGF-beta has a role in determining tumor pathology by regulating myoepithelial or squamous differentiation, maintenance, or transformation.

Conditional deletion of the TGF-beta type II receptor in Col2a expressing cells results in defects in the axial skeleton without alterations in chondrocyte differentiation or embryonic development of long bones.

Members of the TGF-beta superfamily are secreted signaling proteins that regulate many aspects of development including growth and differentiation in skeletal tissue. There are three isoforms of TGF-beta that act through the same heteromeric receptor complex. To address the question of the role of TGF-beta signaling in skeletal development, we generated mice with a conditional deletion of the TGF-beta type II receptor gene (Tgfbr2) specifically in Col2a expressing cells using the Cre/lox recombinase system. Alizarin red-/Alcian blue-stained skeletons were prepared from embryos at 17.5, 15.5, and 13.5 days of gestation. Col2acre+/-;TgfbrloxP/loxP and Col2acre-/-;Tgfbr2+/loxP skeletons were compared. Multiple defects were observed in the base of the skull and in the vertebrae. Specifically, the size and spacing of the vertebrae were altered, and defects were detected in the closure of the neural arches. In addition, alterations in transverse processes, costal joints, and zygapophyses were detected. While the vertebral bodies were only moderately affected, the intervertebral discs (IVDs) were either missing or incomplete. Alterations in the vertebrae could be detected as early as E13.5 days. Surprisingly, alterations in length and mineralization of long bones were not detected at E17.5 days. In addition, the expression patterns of markers for chondrocyte differentiation were not altered in vertebrae or long bones suggesting that loss of responsiveness to TGF-beta in chondrocytes does not affect embryonic endochondral bone formation. In contrast, mice that survived postnatally demonstrated alterations in the length of specific bones. Skeletons from Col2acre+/-;Tgfbr2loxP/loxP mice were compared to those from mice null for the TGF-beta2 ligand. The differences observed between these models allow distinctions to be made between the roles of the various isoforms of TGF-beta and the signaling in specific cell types. The data provide information regarding mechanisms of skeletal development and suggest that TGF-beta signaling is a critical component.

Interferon regulatory factor-7 synergizes with other transcription factors through multiple interactions with p300/CBP coactivators.

Interferon regulatory factor (IRF)-7 is activated in response to virus infection and stimulates the transcription of a set of cellular genes involved in host antiviral defense. The mechanism by which IRF-7 is activated and cooperates with other transcription factors is not fully elucidated. Activation of IRF-7 results from a conformational change triggered by the virus-dependent phosphorylation of its C terminus. This conformational change leads to dimerization, nuclear accumulation, DNA-binding, and transcriptional transactivation. Here we show that activation of IRF-7, like that of IRF-3, is dependent on modifications of two distinct sets of Ser/Thr residues. Moreover, we show that different virus-inducible cis-acting elements display requirements for specific IRFs. In particular, the virus-responsive element of the ISG15 gene promoter can be activated by either IRF-3 or IRF-7 alone, whereas the P31 element of the interferon-beta gene is robustly activated only when IRF-3, IRF-7, and the p300/CBP coactivators are all present. Furthermore, we find that IRF-7 interacts with four distinct regions of p300/CBP. These interactions not only stimulate the intrinsic transcriptional activity of IRF-7, but they are also indispensable for its ability to strongly synergize with other transcription factors, including c-Jun and IRF-3.

Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein-protein interactions.

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3.