Role of Serum Amyloid A as an Intermediate in the IL-1 and PMA-Stimulated Signaling Pathways Regulating Expression of Rabbit Fibroblast Collagenase

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1α. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1α gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1α they can express SAA3, and this autocrine mediator acts independently of IL-1α to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1α to control tissue remodelingin vivo.     

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1^−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.     

mcl-1 Is an Immediate-Early Gene Activated by the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Signaling Pathway and Is One Component of the GM-CSF Viability Response

mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor β chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between −197 and −69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.     

8p12 Stem Cell Myeloproliferative Disorder: the FOP-Fibroblast Growth Factor Receptor 1 Fusion Protein of the t(6;8) Translocation Induces Cell Survival Mediated by Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt/mTOR Pathways

The FOP-fibroblast growth factor receptor 1 (FGFR1) fusion protein is expressed as a consequence of a t(6;8) (q27;p12) translocation associated with a stem cell myeloproliferative disorder with lymphoma, myeloid hyperplasia and eosinophilia. In the present report, we show that the fusion of the leucine-rich N-terminal region of FOP to the catalytic domain of FGFR1 results in conversion of murine hematopoietic cell line Ba/F3 to factor-independent cell survival via an antiapoptotic effect. This survival effect is dependent upon the constitutive tyrosine phosphorylation of FOP-FGFR1. Phosphorylation of STAT1 and of STAT3, but not STAT5, is observed in cells expressing FOP-FGFR1. The survival function of FOP-FGFR1 is abrogated by mutation of the phospholipase C gamma binding site. Mitogen-activated protein kinase (MAPK) is also activated in FOP-FGFR1-expressing cells and confers cytokine-independent survival to hematopoietic cells. These results demonstrate that FOP-FGFR1 is capable of protecting cells from apoptosis by using the same effectors as the wild-type FGFR1. Furthermore, we show that FOP-FGFR1 phosphorylates phosphatidylinositol 3 (PI3)-kinase and AKT and that specific inhibitors of PI3-kinase impair its ability to promote cell survival. In addition, FOP-FGFR1-expressing cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase; this phosphorylation is inhibited by PI3-kinase and mTOR (mammalian target of rapamycin) inhibitors. These results indicate that translation control is important to mediate the cell survival effect induced by FOP-FGFR1. Finally, FOP-FGFR1 protects cells from apoptosis by survival signals including BCL2 overexpression and inactivation of caspase-9 activity. Elucidation of signaling events downstream of FOP-FGFR1 constitutive activation provides insight into the mechanism of leukemogenesis mediated by this oncogenic fusion protein.     

Engagement of the Small GTPase Rab31 Protein and Its Effector,Early Endosome Antigen 1, Is Important for Trafficking of the Ligand-bound Epidermal Growth Factor Receptor from the Early to the Late Endosome

Rab31 is a member of the Rab5 subfamily of Rab GTPases. Although localized largely to the trans-Golgi network, it shares common guanine nucleotide exchange factors and effectors with other Rab5 subfamily members that have been implicated in endocytic membrane traffic. We investigated whether Rab31 also has a role in the trafficking of the ligand-bound EGF receptor (EGFR) internalized through receptor-mediated endocytosis. We found that loss of Rab31 inhibits, but overexpression enhances, EGFR trafficking to the late endosomes and that the effect of Rab31 silencing could be specifically rescued by overexpression of a silencing-resistant form of Rab31. Rab31 was found to interact with the EGFR by coimmunoprecipitation and affinity pulldown analyses, and the primarily trans-Golgi network-localized Rab31 has increased colocalization with the EGFR in A431 cells 30 min after pulsing with EGF. A glycerol gradient sedimentation assay suggested that Rab31 is sequestered into a high molecular weight complex after stimulation with EGF, as was early endosome antigen 1 (EEA1), a factor responsible for endosomal tethering and fusion events. We found that loss of EEA1 reduced the interaction between Rab31 and the EGFR and abrogated the effect of Rab31 overexpression on the trafficking of the EGFR. Likewise, loss of GAPex5, a Rab31 guanine nucleotide exchange factor that has a role in ubiquitination and degradation of the EGFR, reduced the interaction of Rab31 with the EGFR and its effect on EGFR trafficking. Taken together, our results suggest that Rab31 is an important regulator of endocytic trafficking of the EGFR and functions in an EGFR trafficking complex that includes EEA1 and GAPex5.     

MG160, a Membrane Protein of the Golgi Apparatus Which Is Homologous to a Fibroblast Growth Factor Receptor and to a Ligand for E-selectin, Is Found Only in the Golgi Apparatus and Appears Early in Chicken Embryo Development

While over 20 intrinsic proteins of the Golgi apparatus have been identified and sequenced, there is no information on their developmental history, i.e., whether all Golgi proteins are expressed simultaneously or whether there is a hierarchical or stage-specific order of their expression during embryonic development. In this study we have examined the emergence and distribution of MG160 during the development of chicken embryos. MG160 is a conserved membrane sialoglycoprotein of the Golgi apparatus of most cells displaying over 90% amino acid sequence identities with two apparently unrelated molecules, namely CFR, a chicken fibroblast growth factor receptor, and ESL-1, a ligand for E-selectin (Gonatas et al., J. Biol. Chem. 1989, 264, 646-653; Burrus and Olwin, J. Biol. Chem. 1989, 264, 18647-18653; Burrus et al., Mol. Cell Biol. 1992, 12, 5600-5609; Gonatas et al., J. Cell Sci. 108, 457-467; Steegmaier et al., Nature 1995, 373, 615-620). This study was carried out by in situ hybridization, using a 56-mer antisense probe for the chicken homologue of MG160 which differs only by four bases from the corresponding segment of the rat cDNA and by immunocytochemistry and Western blotting using a polyclonal antiserum against MG160. The protein was ubiquitously and exclusively localized in the Golgi apparatus and appeared early in development within the ectoblast and primitive endoblast prior to the formation of the primitive streak. At 2 to 3 days, MG160 was particularly prominent in the notochord, neural tube, somites, and cartilage cells. In organs with central lumens, such as the neural tube, the Golgi apparatus, visualized by immunostaining for MG160, was elongated and it was located at the apical pole of cells. In 6-day-old embryos, the ongoing physiologic degeneration of the notochord was accompanied by fragmentation of the immunostained Golgi apparatus and decreased labeling of the mRNA for MG160. In order to gain information on possible interactions between MG160 and basic fibroblast growth factor (bFGF), the localization of both molecules was studied by immunocytochemistry in 3-day-old chicken embryos. While MG160 was ubiquitous in the Golgi apparatus of all cells and tissues, endogenous bFGF was not detected while exogenous bFGF bound only to basement membranes. These results indicate that MG160 is a primordial protein of the Golgi apparatus and are consistent with the hypothesis that the binding of MG160 to fibroblast growth factors and E-selectin is not related to the still unknown principal function of MG160 in the Golgi apparatus.