AKT Signaling Downstream of KGF Is Necessary and Sufficient for Blocking Cyclophosphamide Bladder Injury.

Keratinocyte growth factor (KGF) drives phosphorylated (activated) AKT (pAKT) in bladder urothelium, which correlates with cytoprotection from cyclophosphamide. The current study determined whether: i) KGF modifies AKT targets [B-cell lymphoma protein 2-associated agonist of cell death (BAD) and mammalian target of rapamycin complex (mTORC)-1] that could block apoptosis; ii) AKT signaling is required for KGF cytoprotection; iii) direct AKT activation drives cytoprotection; iv) co-administration of KGF and an AKT inhibitor blocks urothelial cytoprotection and AKT and AKT-target activation; and v) an AKT agonist prevents cyclophosphamide-induced urothelial apoptosis. Mice were given KGF and cyclophosphamide (or sham injury), and pBAD (readout of BAD inhibition) or p-p70S6k (pS6, readout of mTORC1 signaling) was assessed. KGF induced pBAD urothelial staining and prevented cyclophosphamide-induced loss of urothelial pS6 staining (likely stabilizing mTORC1 activity). Co-administration of KGF and AKT inhibitor blocked KGF-driven urothelial cytoprotection from cyclophosphamide and prevented pAKT, pBAD, and pS6 urothelial expression. Conversely, systemic AKT agonist blocked cyclophosphamide-induced urothelial apoptosis and induced pAKT, pBAD, and pS6, similar to KGF. Thus, the KGF-AKT signaling axis appeared to phosphorylate (suppress) BAD and prevent cyclophosphamide-induced loss of mTORC1 signaling, both of which likely suppress apoptosis. Additionally, AKT signaling was required for KGF-driven cytoprotection, and direct AKT activation was sufficient for blocking apoptosis. Thus, AKT may be a therapeutic target for blocking urothelial apoptosis from cyclophosphamide.

Loss of Fibroblast Growth Factor Receptor 2 (FGFR2) Leads to Defective Bladder Urothelial Regeneration after Cyclophosphamide Injury.

Cyclophosphamide may cause hemorrhagic cystitis and eventually bladder urothelial cancer. Genetic determinants for poor outcomes are unknown. We assessed actions of fibroblast growth factor receptor (FGFR) 2 in urothelium after cyclophosphamide exposure. Conditional urothelial deletion of Fgfr2 (Fgfr2KO) did not affect injury severity or proliferation of keratin 14+ (KRT14+) basal progenitors or other urothelial cells 1 day after cyclophosphamide exposure. Three days after cyclophosphamide exposure, Fgfr2KO urothelium had defective regeneration, fewer cells, larger basal cell bodies and nuclei, paradoxical increases in proliferation markers, and excessive replication stress versus controls. Fgfr2KO mice had evidence of pathologic basal cell endoreplication associated with absent phosphorylated ERK staining and decreased p53 expression versus controls. Mice with conditional deletion of Fgfr2 in urothelium enriched for KRT14+ cells reproduced Fgfr2KO abnormalities after cyclophosphamide exposure. Fgfr2KO urothelium had defects up to 6 months after injury versus controls, including larger basal cells and nuclei, more persistent basal and ectopic lumenal KRT14+ cells, and signs of metaplasia (attenuated E-cadherin staining). Mice missing one allele of Fgfr2 also had (less severe) regeneration defects and basal cell endoreplication 3 days after cyclophosphamide exposure versus controls. Thus, reduced FGFR2/ERK signaling apparently leads to abnormal urothelial repair after cyclophosphamide exposure from pathologic basal cell endoreplication. Patients with genetic variants in FGFR2 or its ligands may have increased risks of hemorrhagic cystitis or urothelial cancer from persistent and ectopic KRT14+ cells.

Keratinocyte Growth Factor Reduces Injury and Leads to Early Recovery from Cyclophosphamide Bladder Injury.

Keratinocyte growth factor (KGF) improves cyclophosphamide-induced bladder injury. To understand the mechanisms, we subcutaneously administered KGF to mice 24 hours before i.p. cyclophosphamide administration, followed by histologic assays and immunostaining. In vehicle (phosphate-buffered saline)-pretreated mice, nonapoptotic superficial cell death from 2 to 6 hours and apoptosis in intermediate and basal cells from 4 to 24 hours was observed after cyclophosphamide. Despite superficial cell loss, KGF suppressed intermediate and basal cell apoptosis, likely via AKT signaling. At 6 and 24 hours after cyclophosphamide, KGF-pretreated mice also had apparent extracellular signal-regulated kinase (ERK)-driven proliferation of mostly keratin 5 (KRT5)+/KRT14- intermediate cells. At 1 to 28 days after cyclophosphamide treatment, mostly KRT14+ basal progenitor cells proliferated in response to injury, peaking at 3 days in both treatment groups; however, proliferation rates were lower in the KGF group at 3 days, consistent with less injury. Three days after injury, unlike controls, KGF-pretreated mice had regenerated superficial cells. At 10 and 28 days after cyclophosphamide treatment, KGF-pretreated mice had little proliferation and marked restoration of urothelial layers, whereas the phosphate-buffered saline group had ongoing regeneration. Administration of KGF to uninjured mice reproduced ERK-driven KRT5+/KRT14- proliferation seen in injured mice; KRT14+ cells were unaffected. KGF pretreatment blocks cyclophosphamide-induced intermediate and basal cell apoptosis, likely by phosphorylated AKT, and drives phosphorylated ERK-mediated KRT5+/KRT14- cell proliferation, leading to early urothelial regeneration.

Loss of peri-Wolffian duct stromal Frs2α expression in mice leads to abnormal ureteric bud induction and vesicoureteral reflux.

BackgroundFibroblast growth factor receptor 2 (Fgfr2) deletion from murine peri-Wolffian duct stroma (ST) results in aberrant ureteric bud induction, abnormal ureteral insertion into the bladder, and high rates of vesicoureteral reflux (VUR). It is unclear which receptor docking protein(s) is/are responsible for Fgfr2 actions in these tissues. We investigated whether the docking protein, fibroblast receptor substrate 2α (Frs2α), had a role in peri-Wolffian duct ST similar to Fgfr2.MethodsWe conditionally deleted Frs2α in peri-Wolffian duct ST with a Tbx18cre mouse line (Frs2αST-/-). We assessed for ureteric induction defects and alterations in downstream targets mediating defects. We performed euthanized cystograms and assessed ureter-bladder junctions by three-dimensional (3D) reconstructions.ResultsEmbryonic day (E) 11.5 Frs2αST-/- embryos had many displaced ureteric bud induction sites when compared with controls. E11.0 Frs2αST-/- embryos had decreased Bmp4 expression and signaling, which can cause abnormal ureteric bud induction. Postnatal day 1 (P1) and P30 Frs2αST-/- mice had higher VUR rates and grades vs. CONTROLS: Mutant refluxing ureters that inserted improperly into the bladder had shortened intravesicular tunnels (IVTs) when compared with controlsConclusionFrs2αST-/- embryos have aberrant ureteric induction sites, improper ureteral insertion, shortened intravesicular lengths, and VUR. Induction site defects appear secondary to reduced Bmp4 expression, similar to Fgfr2 mutants.

Follistatin-like protein 1 regulates chondrocyte proliferation and chondrogenic differentiation of mesenchymal stem cells.

OBJECTIVES: Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. METHODS: To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. RESULTS: The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor ß signalling in FSTL1 KO cells was significantly suppressed. CONCLUSIONS: FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.

Dicer function is required in the metanephric mesenchyme for early kidney development.

MicroRNAs (miRNAs) are small, noncoding regulatory RNAs that act as posttranscriptional repressors by binding to the 3'-untranslated region (3'-UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at midgestation (Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA. J Am Soc Nephrol 22: 1053-1063, 2011). To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1, and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the proapoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis and indicate a crucial role for miRNAs in regulating the survival of this lineage.

FSTL1 promotes arthritis in mice by enhancing inflammatory cytokine/chemokine expression.

OBJECTIVE: FSTL1 is a secreted glycoprotein that exacerbates murine arthritis and is overexpressed in human arthritis. The aim of this study was to determine the mechanism by which FSTL1 promotes arthritis. METHODS: Collagen-induced arthritis was induced in mice hypomorphic for FSTL1, generated with a gene trap-targeted mutant embryonic stem cell line. Arthritis was assessed by measuring paw swelling and using a qualitative arthritis index. Bone marrow-derived mesenchymal stromal cells from hypomorphic mice, as well as mouse stromal ST2 cells transduced with short hairpin RNA to suppress FSTL1 expression, were stimulated with interleukin-1ß (IL-1ß), tumor necrosis factor α, and IL-17. The monocyte cell line U937, which does not express FSTL1, was transfected with a plasmid encoding FSTL1 and stimulated with phorbol myristate acetate and lipopolysaccharide. Cell supernatants were assayed for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and FSTL1 by enzyme-linked immunosorbent assay. RESULTS: FSTL1 hypomorphic mice had reduced levels of FSTL1 compared to littermate controls. Following induction of arthritis, a significant correlation was observed between serum FSTL1 levels and both paw swelling and the arthritis index. Similar correlations were observed between the amount of FSTL1 produced by mesenchymal stromal cells, stromal ST2 cells, and monocytes and the secretion of IL-6, IL-8, and MCP-1. CONCLUSION: These findings demonstrate that FSTL1 up-regulates proinflammatory mediators important in the pathology of arthritis, and that serum levels of FSTL1 correlate with severity of arthritis. The latter supports the possibility that FSTL1 might be a target for treatment of certain forms of arthritis.

Follistatin-like protein 1 is a mesenchyme-derived inflammatory protein and may represent a biomarker for systemic-onset juvenile rheumatoid arthritis.

OBJECTIVE: To examine both the source of follistatin-like protein 1 (FSTL-1) and the factors that induce its expression in arthritis, and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by overexpression of FSTL-1. METHODS: FSTL-1 expression patterns were analyzed by immunohistochemical staining of joint tissue derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to transforming growth factor beta (TGFbeta), interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-6. In addition, sera and synovial fluid from children with oligoarticular, polyarticular, or systemic-onset JRA were assayed for FSTL-1 using a custom enzyme-linked immunosorbent assay. FSTL-1 concentrations in these patients were assessed for correlations with the erythrocyte sedimentation rate (ESR) and platelet count. RESULTS: Immunohistochemical staining of murine joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGFbeta, IL-1beta, TNFalpha, and IL-6. The IL-1beta response was significantly greater than the TNFalpha response (P < 0.05). In human serum and synovial fluid, only those samples from children with the systemic-onset JRA subtype had elevated concentrations of FSTL-1. The synovial fluid concentrations of FSTL-1 were 2-3-fold higher than the serum concentrations. The elevation in serum FSTL-1 concentrations seen in children with systemic-onset JRA correlated closely with elevations in the ESR and platelet count. CONCLUSION: These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1beta is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA.