BMP-induced non-canonical signaling is upregulated during autophagy-mediated regeneration in inflamed mesothelial cells.

Previously, we showed that after Freund's adjuvant-induced peritonitis, rat mesothelial cells regain their epithelial phenotype through mesenchymal-epithelial transition (MET) accompanied by autophagy. Since bone morphogenetic proteins (BMPs) are well-known MET-inducers, we were interested in the potential expression of BMPs and BMP-induced pathways. Although mesothelial cells expressed lower amounts of BMP7, its level in the peritoneal cavity and mesothelial synthesis of BMP4 were significantly increased during inflammation. BMPR1A and BMPR2 were also significantly expressed. Expression of transforming growth factor beta-activated kinase (TAK1) and c-Jun NH2-terminal kinases (JNK1-JNK2) were more intense than that of phosphorylated Mothers Against Decapentaplegic homolog 1/5 (p-SMAD1/5), confirming that the non-canonical pathway of BMPs prevailed in our model. JNK signaling through B-cell lymphoma-2 (Bcl-2) can contribute to Beclin-1 activation. We demonstrated that TAK1-JNK-Bcl-2 signaling was upregulated simultaneously with the autophagy-mediated regeneration. A further goal of our study was to prove the regenerative role of autophagy after inflammation. We used a specific inhibitor, bafilomycin A1 (BafA1), and found that BafA1 treatment decreased the expression of microtubule-associated protein 1A/1B-light chain 3 (LC3B) and resulted in morphological signs of cell death in inflamed mesothelial cells indicating that if autophagy is arrested, regeneration turns into cell death and consequently, mesothelial cells die.

Endocytosis of GM-CSF receptor ß is essential for signal transduction regulating mesothelial-macrophage transition.

During Freund's adjuvant induced inflammation rat mesenteric mesothelial cells transdifferentiate into mesenchymal cell. They express macrophage markers, inflammatory cytokines (TGF-ß, TNFα, IL-6), and specific receptors. When primary mesenteric cultures were treated with GM-CSF and/or TGF-ß (in vitro), similar phenotypic and biological changes were induced. It seemed likely that GM-CSF receptor-ligand complex should be internalized to initiate mesothelial-macrophage transition. To follow the intracellular route of GM-CSF receptor ß, we co-localized this receptor with various endocytic markers (Cav-1, EEA1, Rab7, and Rab11a), and carried out detailed immunocytochemical, statistical and biochemical analyses. Since STAT5 is one of the downstream element of GM-CSF signaling, we followed the expression and phosphorylation level of this transcription factor. Our results showed that in mesenteric mesothelial cells GM-CSF receptor ß is internalized by caveolae, delivered into early endosomes where the signaling events occur, STAT5A is phosphorylated by JAK2, and then translocated into the nucleus. When dynamin-dependent endocytosis of GM-CSFR ß is inhibited by dynasore, phosphorylation of STAT5A is not occurred, confirming, that the internalization of receptor ß is indispensable for signal transduction. At the early time of inflammation a significant receptor recycling can be found to the plasma membrane. Later (day 8) the receptor is delivered into late endosomes, indicating that its degradation has already started, and the regeneration of mesothelial cells can start. All of these data strongly support that the internalization of GM-CSF receptor ß is required and essential for signal transduction.

Inflammation-Induced Epithelial-to-Mesenchymal Transition and GM-CSF Treatment Stimulate Mesenteric Mesothelial Cells to Transdifferentiate into Macrophages.

In our previous work, we showed that during inflammation-induced epithelial-to-mesenchymal transition (EMT), mesenteric mesothelial cells express ED1 (pan-macrophage marker), indicating that they are transformed into macrophage-like cells. In this paper, we provide additional evidences about this transition by following the phagocytic activity and the TNFα production of mesenteric mesothelial cells during inflammation. Upon injection of India ink particles or fluorescent-labeled bioparticles (pHrodo) into the peritoneal cavity of rats pretreated with Freund's adjuvant, we found that mesothelial cells efficiently engulfed these particles. A similar increase of internalization could be observed by mesothelial cells in GM-CSF pretreated primary mesenteric culture. Since macrophages are the major producers of tumor necrosis factor, TNFα, we investigated expression level of TNFα during inflammation-induced EMT and found that TNFα was indeed expressed in these cells, reaching the highest level at the 5th day of inflammation. Since TNFα is one of the target genes of early growth response (EGR1) transcription factor, playing important role in monocyte-macrophage differentiation, expression of EGR1 in mesothelial cells was also investigated by Western blot and immunocytochemistry. While mesothelial cells did not express EGR1, a marked increase was observed in mesothelial cells by the time of inflammation. Parallel to this, nuclear translocation of EGR1 was shown by immunocytochemistry at the day 5 of inflammation. Caveolin-1 level was high and ERK1/2 became phosphorylated as the inflammation proceeded showing a slight decrease when the regeneration started. Our present data support the idea that under special stimuli, mesenteric mesothelial cells are able to transdifferentiate into macrophages, and this transition is regulated by the caveolin-1/ERK1/2/EGR1 signaling pathway.

Autophagy is the key process in the re-establishment of the epitheloid phenotype during mesenchymal-epithelial transition (MET).

In previous studies we showed that during Freund's adjuvant induced inflammation rat mesenteric mesothelial cells undergo epithelial-mesenchymal transition type II (EMT). This process was characterized by a dramatic increase of the number of cell organelles and volume of mesothelial cells. After the inflammation reached its maximum, the mesenchymal-like cells gradually regained their epithelial phenotype (mesenchymal-epithelial transition, MET). During the recovery process, the decrease of the number of cell organelles was accompanied by an increasing number of autophagic structures in the cytoplasm, indicating that autophagy might play crucial role in MET. Morphometric data of this study showed that the number of the autophagic organelles increased by the time of inflammation and was the highest at day 7-8, when regeneration started. These morphological observations were supported by immunocytochemistry and Western blot analyses with various markers, directly or indirectly involved in this process. Endocytic markers were expressed at high level during both EMT and MET, while the expression of factors regulating autophagy simultaneously changed with the morphology: p-Akt and p-mTOR level was high at day 3-5 and significantly decreased when autophagy speeded up. The Beclin-1, which is the key factor of initiating autophagy, was expressed at the early time of inflammation. These results strongly suggest that autophagy plays important role in regeneration (MET), and it is regulated and synchronized by various signalling events during inflammation.

GM-CSF and GM-CSF receptor have regulatory role in transforming rat mesenteric mesothelial cells into macrophage-like cells.

OBJECTIVE AND DESIGN: During peritonitis, mesothelial cells assume macrophage characteristics, expressing macrophage markers, indicating that they might differentiate into macrophage-like cells. MATERIALS AND SUBJECTS: Twenty-five male rats were used for in vivo experiments. For in vitro experiments, a primary mesentery culture model was developed. The mesothelial cell to macrophage-like cell transition was followed by studying ED1 expression. TREATMENTS: In vitro primary mesenteric culture was treated with granulocyte-macrophage colony-stimulating factor (GM-CSF, 1 ng/ml). Blocking internalization of receptor-ligand complex, Dynasore (80 µM) was used. Acute peritonitis was induced by Freund's adjuvant's (1 ml) intraperitoneal injection. RESULTS: Immunohistochemistry: GM-CSF in vitro treatment resulted in a prominent ED1 expression in transformed mesothelial cells. Blocking the internalization, ED1 expression could not be detected. GM-CSF receptor (both α and ß) was expressed in mesothelial cells in vitro (even if the GM-CSF was not present) and in vivo. Inflammation resulted in an increasing GM-CSF and GM-CSF-receptor level in the lysate of mesothelial cells. CONCLUSIONS: Mesothelial cells can differentiate into macrophage-like cells, and GM-CSF, produced by the mesothelial cells, has probably an autocrine regulatory role in this transition. Our results provide new data about the plasticity of mesothelial cell and support the idea that during inflammation macrophages can derive from non-hematopoietic sources as well.