ABSTRACT
Epithelial injury is a key initiator of fibrosis but - in contrast to the previous paradigm - the epithelium in situ does not undergo wide-spread epithelial-mesenchymal/myofibroblast transition (EMT/EMyT). Instead, it assumes a Profibrotic Epithelial Phenotype (PEP) characterized by fibrogenic cytokine production. The transcriptional mechanisms underlying PEP are undefined. As we have shown that two RhoA/cytoskeleton-regulated transcriptional coactivators, Myocardin-related transcription factor (MRTF) and TAZ, are indispensable for EMyT, we asked if they might mediate PEP as well. Here we show that mechanical stress (cyclic stretch) increased the expression of transforming growth factor-ß1 (TGFß1), connective tissue growth factor (CTGF), platelet-derived growth factor and Indian Hedgehog mRNA in LLC-PK1 tubular cells. These responses were mitigated by siRNA-mediated silencing or pharmacological inhibition of MRTF (CCG-1423) or TAZ (verteporfin). RhoA inhibition exerted similar effects. Unilateral ureteral obstruction, a murine model of mechanically-triggered kidney fibrosis, induced tubular RhoA activation along with overexpression/nuclear accumulation of MRTF and TAZ, and increased transcription of the above-mentioned cytokines. Laser capture microdissection revealed TAZ, TGFß1 and CTGF induction specifically in the tubular epithelium. CCG-1423 suppressed total renal and tubular expression of these proteins. Thus, MRTF regulates epithelial TAZ expression, and both MRTF and TAZ are critical mediators of PEP-related epithelial cytokine production.
Subject(s)
Epithelial Cells/pathology , Fibrosis/pathology , Trans-Activators/physiology , Transcription Factors/physiology , Adaptor Proteins, Signal Transducing , Animals , Cytokines/metabolism , Kidney/metabolism , Mice , Stress, Mechanical , Trans-Activators/metabolism , Transcription Factors/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk(-/-) mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk(-/-) HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk(-/-) HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk.
Subject(s)
Gamma Rays/adverse effects , Interleukin-11/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Leukemia, B-Cell/metabolism , MAP Kinase Signaling System/radiation effects , Neoplasm Proteins/metabolism , Neoplasms, Radiation-Induced/metabolism , Proteins/metabolism , Radiation Tolerance/radiation effects , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Animals , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/metabolism , Humans , Interleukin-11/genetics , Intracellular Signaling Peptides and Proteins/genetics , Leukemia, B-Cell/genetics , Leukemia, B-Cell/pathology , MAP Kinase Signaling System/genetics , Membrane Proteins , Mice , Mice, Knockout , Neoplasm Proteins/genetics , Neoplasms, Radiation-Induced/genetics , Neoplasms, Radiation-Induced/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proteins/genetics , Radiation Tolerance/geneticsABSTRACT
GYF domains are small, versatile adaptor domains that recognize proline-rich sequences (PRS). They are present in most eukaryotic species sequenced so far, but in contrast to other PRS-recognition domains (PRD), GYF domains have not experienced the same amplification in metazoa during evolution. Mutational and structural analysis has shown the conserved signature W-X-Y-X(6-11)-GPF-X(4)-M-X(2)-W-X(3)-GYF to be the site of interaction with proline-rich peptides. In contrast, composition and length of the C-terminal half of GYF domains are not conserved. Similar to other PRD, GYF domains bind to many different PRS that converge on a minimal consensus sequence. All GYF domains analyzed so far selected for the core motif PPG, whereas amino-acid preferences adjacent to this motif vary. As a result of this analysis, two subfamilies have been identified: CD2BP2-type and SMY2-type GYF domains. The latter subfamily comprises most GYF domains and is characterized by a shorter beta(1)-beta(2) loop and an aspartate instead of the tryptophan found at position 8 in CD2BP2-type GYF domains. Recent analysis of binding specificities for GYF domains allowed identification of novel interaction partners. Thereby proteomics has contributed to a functional understanding of GYF domain-containing proteins and sets the stage for a more systematic investigation of their functions in vivo.
