Transcriptomic Profiling of DAF-7/TGFß Pathway Mutants in C. elegans.

The transforming growth factor beta superfamily encompasses a large family of ligands that are well conserved across many organisms. They are regulators of a number of physiological and pathological processes. The model nematode Caenorhabditis elegans has been instrumental in identifying key components of the transforming growth factor beta (TGFß) pathway. In C. elegans, the TGFß homolog DAF-7 signals through the DAF-1 Type I and DAF-4 Type II receptors to phosphorylate downstream R-SMADs DAF-8 and DAF-14. These R-SMADs translocate into the nucleus to inhibit Co-SMAD DAF-3. Many of the roles of the canonical DAF-7 pathway, involving both DAF-1 and DAF-3, have been identified using targeted genetic studies. Few have assessed the global transcriptomic changes in response to these genes, especially in adult animals. In this study, we performed RNA sequencing on wild type, daf-1, and daf-1; daf-3 adult hermaphrodites. To assess the overall trends of the data, we identified differentially expressed genes (DEGs) and performed gene ontology analysis to identify the types of downstream genes that are differentially expressed. Hierarchical clustering showed that the daf-1; daf-3 double mutants are transcriptionally more similar to wild type than daf-1 mutants. Analysis of the DEGs showed a disproportionally high number of genes whose expression is increased in daf-1 mutants, suggesting that DAF-1 acts as a general repressor of gene expression in wild type animals. Gene ontology analysis of the DEGs produced many significantly enriched terms, including Molting Cycle, Response to Topologically Incorrect Protein, and Response to Biotic Stimulus. Understanding the direct and indirect targets of the DAF-7 TGFß pathway through this RNA-seq dataset can provide insight into novel roles of the multifunctional signaling pathway, as well as identify novel genes that may participate in previously reported functions of TGFß signaling.

Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton.

Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes.

Prolactin and transforming growth factor-beta signaling exert opposing effects on mammary gland morphogenesis, involution, and the Akt-forkhead pathway.

Both prolactin (PRL) and TGF-beta regulate cell survival in mammary epithelial cells, but their mechanisms of interactions are not known. In primary mammary epithelial cells and the HC11 mouse mammary epithelial cell line, PRL prevented TGF-beta-induced apoptosis, as measured by terminal deoxynucleotidyltransferase dUTP nick-end labeling staining and caspase-3 activation. This effect depended on phosphatidyl inositol triphosphate kinase (PI3K). PI3K activates a downstream serine/threonine kinase, Akt; therefore, we investigated the role of Akt in the interaction between PRL and TGF-beta signaling. Akt activity was inhibited by TGF-beta over a 20- to 60-min time course. In TGF-beta-treated cells, PRL disinhibited Akt in a PI3K-dependent manner. Expression of dominant negative Akt blocked the protective effect of PRL in TGF-beta-induced apoptosis. Transgenic mice overexpressing a dominant-negative TGF-beta type II receptor (DNIIR) in the mammary epithelium undergo hyperplastic alveolar development, and this effect was PRL dependent. Involution in response to teat sealing was slowed by overexpression of DNIIR; furthermore, Akt and forkhead phosphorylation increased in the sealed mammary glands of DNIIR mice. Thus, Akt appears to be an essential component of the interaction between PRL and TGF-beta signaling in mammary epithelial cells both in vitro and in vivo.