Abstract
In the hematopoietic system, TGF-β1 is one of the most potent extrinsic regulators, affecting both early progenitors and committed cells. At the top of the hematopoietic hierarchy, TGF-β1 maintains hematopoietic stem cells (HSCs) in quiescence in vitro through transcriptional regulation of genes encoding proteins important in the cell cycle. We have shown that TGF-β receptor I (TβRI) −/− HSCs exhibit increased proliferative capacity in vitro and that TβRII−/− mice develop a multifocal autoimmune disease, mainly mediated by T-cells (Larsson et al, 2003, Levéen et al 2002). The mechanisms of TGF-β signaling in hematopoietic cells are poorly understood and many target genes of TGF-β signaling remain elusive. In this study we have used global gene expression analysis to investigate whether all TGF-β signaling is mediated by TβRI and II. Furthermore, we asked what target genes are affected upon TGF-β stimulation in normal and TGF-β signaling deficient murine embryonic fibroblasts (MEFs). MEFs were grown with and without TGF-β1 stimulation and proliferation, transcriptional responses and expression analysis were performed. We demonstrate through Western Blot analysis, luciferase reporter assays and cell expansion experiments how these cells lack functional TβRI. Additionally, transcriptional assays show that no other Smad activity is triggered by TGF-β1 stimulation. Furthermore, we demonstrate through quantitative RT-PCR that the inhibitor of differentiation family of genes, known targets of TGF-β signaling, are not affected by TGF-β1 in TβRI−/− MEFs, while wt cells downregulate these genes 4–8.5 fold in response to stimulation. In order to completely exclude alternative receptors outside the TGF-β superfamily and signaling pathways activated through TβRII alone, we performed global gene expression profiling on TGF-β1 stimulated TβRI−/− MEFs with unstimulated TβRI deficient cells as reference. Very few (0.05 %) of the more than 37,000 spots on the microarray had a >2 fold differential expression in the two experiments conducted. Similar experiments performed on wt cells resulted in differential expression of between 2.6–3.9 % of the genes printed. From this data we conclude that no signaling affecting gene expression occur in the absence of TβRI in these cells. Additionally we present transcriptional profiles of MEF cell lines that either are normal or are TβRI deficient. By means of cDNA microarray technology, we have identified genes that were differentially expressed when TβRI deficient fibroblasts were compared to wt cells stimulated with TGF-β1. Our results create a data base of 461 significantly differentially expressed (p<0.01) target genes of TGF-β signaling. These include genes potentially responsible for the growth arrest induced by TGF-β1, like Gadd45g, Gas5, Id1, Id2 and Id3. However, the most significantly enriched number of differentially expressed genes are involved in protein folding and chaperone activities (Hspa9a, Hsp105, Hspe1, Hsp60, Cct2, Cct3, Cct8, Tcp1 and Dnaja1. Studies to identify TGF-β signaling responsive genes in HSCs are in progress.
Hierarchy of TGFβ/SMAD, Hippo/YAP/TAZ, and Wnt/β-catenin signaling in melanoma phenotype switching
