Cyclosporine A induces apoptotic and autophagic cell death in rat pituitary GH3 cells.

Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the development of chronic nephrotoxicity. In this study, we investigated CsA treatment induced apoptotic and autophagic cell death in pituitary GH3 cells. CsA treatment (0.1 to 10 µM) decreased survival of GH3 cells in a dose-dependent manner. Cell viability decreased significantly with increasing CsA concentrations largely due to an increase in apoptosis, while cell death rates due to autophagy altered only slightly. Several molecular and morphological features correlated with cell death through these distinct pathways. At concentrations ranging from 1.0 to 10 µM, CsA induced a dose-dependent increase in expression of the autophagy markers LC3-I and LC3-II. Immunofluorescence staining revealed markedly increased levels of both LC3 and lysosomal-associated membrane protein 2 (Lamp2), indicating increases in autophagosomes. At the same CsA doses, apoptotic cell death was apparent as indicated by nuclear and DNA fragmentation and increased p53 expression. In apoptotic or autophagic cells, p-ERK levels were highest at 1.0 µM CsA compared to control or other doses. In contrast, Bax levels in both types of cell death were increased in a dose-dependent manner, while Bcl-2 levels showed dose-dependent augmentation in autophagy and were decreased in apoptosis. Manganese superoxide dismutase (Mn-SOD) showed a similar dose-dependent reduction in cells undergoing apoptosis, while levels of the intracellular calcium ion exchange maker calbindin-D9k were decreased in apoptosis (1.0 to 5 µM CsA), but unchanged in autophagy. In conclusion, these results suggest that CsA induction of apoptotic or autophagic cell death in rat pituitary GH3 cells depends on the relative expression of factors and correlates with Bcl-2 and Mn-SOD levels.ope

Lysosomal dysfunction of corneal fibroblasts underlies the pathogenesis of Granular Corneal Dystrophy Type 2 and can be rescued by TFEB

Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor β-induced (TGFBI) gene-linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant-TGFBIp. The defective autophagic degradation of pathogenic mutant-TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro-enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant-TGFBIp. Exogenous CTSL expression dramatically reduced mutant-TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant-TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.ope

Compound heterozygous mutations in TGFBI cause a severe phenotype of granular corneal dystrophy type 2

We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-β-induced (TGFBI) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole-TGFBI. The patient's family was tested to determine the pedigrees. Five novel mutations (p.(His174Asp), p.(Ile247Asn), p.(Tyr88Cys), p.(Arg257Pro), and p.(Tyr468*)) and two known mutations (p.(Asn544Ser) and p.(Arg179*)) in TGFBI were identified, along with p.(Arg124His), in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.(Arg124His) and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.(Ile247Asn) and p.(Asn544Ser)); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.(Ile247Asn)). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype-phenotype correlation and additional care when treating TGFBI corneal dystrophies.ope

Altered mitochondrial function in type 2 granular corneal dystrophy

Type 2 granular corneal dystrophy (GCD2) is caused by point mutation R124H in the transforming growth factor-β-induced gene (TGFBI) and is characterized by age-dependent progression of corneal deposits. Mitochondrial features in heterozygous GCD2 and normal corneal tissues was evaluated using electron microscopy. Primary corneal fibroblasts of homozygous and normal corneas were cultured to passage 4 or 8. Keratocytes of normal corneal tissue are narrow, and details of their intracellular organelles are difficult to distinguish. Keratocytes of heterozygous GCD2 tissues exhibited many degenerative mitochondria. MitoTracker and cytochrome c staining demonstrated increased mitochondrial activity in mutated cells at early passages. Decreases in depolarized mitochondria, cellular proliferation, and expression of complexes I to V and increases in apoptotic change were observed in late-passage mutant fibroblasts. PGC-1α, ANT-1, p-Akt, and p-mTOR but not NF-κB expression demonstrated a passage-dependent decrease in all cells. Increased passage- or mutation-related intracellular reactive oxygen species and delayed proliferation of methanethiosulfonate (MTS) were recovered using application of antioxidant butylated hydroxyanisole. Mitochondrial features and function were altered in mutated GCD2 keratocytes, in particular in older cells. Alteration of mitochondrial function is critical for understanding the pathogenesis of GCD2.ope

Lithium inhibits tumor lymphangiogenesis and metastasis through the inhibition of TGFBIp expression in cancer cells

Metastasis is the main cause of mortality in cancer patients. Although there are many anti-cancer drugs targeting tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression; in particular, lymphangiogenesis is pivotal for metastasis in cancer. Here we report that lithium inhibits colon cancer metastasis by blocking lymphangiogenesis. Lithium reduces the expression of transforming growth factor-β-induced protein (TGFBIp) in colon cancer cells by inhibiting Smad3 phosphorylation via GSK3β inactivation. Moreover, lithium inhibits lymphatic endothelial cell migration, which is increased upon TGFBIp expression in tumor cells. Lithium had no significant effect on SW620 tumor growth in vitro and in vivo; however, it inhibited lymphangiogenesis in tumors. In tumor xenografts model, lithium was found to prevent metastasis to the lungs, liver, and lymph nodes by inhibiting TGFBIp-induced tumor lymphangiogenesis. Collectively, our findings demonstrate a novel role of lithium in the inhibition of colon cancer metastasis by blocking TGFBIp expression, and thereby TGFBIp-induced lymphangiogenesis, in primary tumors.ope

Role of TGFBIp in Wound Healing and Mucin Expression in Corneal Epithelial Cells

PURPOSE: Transforming growth factor-β-induced protein (TGFBIp) is highly expressed in the cornea, and mutant TGFBIp induces corneal diseases. However, the function of TGFBIp in cornea epithelium is not fully investigated. Here, we tested the importance of TGFBIp in regulation of gene expression and corneal epithelial cell (CEC) activity. MATERIALS AND METHODS: The effect of TGFBIp on CEC activity was analyzed by cell migration, adhesion, proliferation and wound healing assay. Analysis of gene expression was examined by western blot and quantitative reverse transcription PCR. RESULTS: The results demonstrated that TGFBIp increased adhesion, migration, proliferation, and wound healing of CECs. Analysis of gene expression presented that TGFBIp-stimulated CECs exhibited increased expression of mucin family genes, such as MUC1, -4, -5AC, and -16. Furthermore, TGFBIp treatment increased the expression of MUC1, -4, -5AC, -7, and -16 in conjunctival epithelial cells. TGFBIp also increased the activity of intracellular signaling molecules ERK and AKT in CECs. Using pharmacologic inhibitors of ERK and AKT, we showed that the expression of mucin genes by TGFBIp is mediated by the activation of ERK and AKT signaling. CONCLUSION: Our findings demonstrate that the locally generated TGFBIp in the cornea may contribute to wound healing of CECs by enhancing the migration, adhesion, and proliferation of CECs. In addition, our results suggest that TGFBIp has a protective effect on ocular surfaces by inducing the expression of mucin genes in corneal and conjunctival epithelial cells. These data suggest that TGFBIp is a useful therapeutic target for patients with corneal wounds.ope

Histone methylation levels correlate with TGFBIp and extracellular matrix gene expression in normal and granular corneal dystrophy type 2 corneal fibroblasts

BACKGROUND: TGFβ1-induced expression of transforming growth factor β-induced protein (TGFBIp) and extracellular matrix (ECM) genes plays a major role in the development of granular corneal dystrophy type 2 (GCD2: also called Avellino corneal dystrophy). Although some key transcription factors are known, the epigenetic mechanisms modulating TGFBIp and ECM expression remain unclear. We examined the role of chromatin markers such as histone H3 lysine methylation (H3Kme) in TGFβ1-induced TGFBIp and ECM gene expression in normal and GCD2-derived human corneal fibroblasts. METHODS: Wild-type (n = 3), GCD2-heterozygous (n = 1), and GCD2-homozygous (n = 3) primary human corneal fibroblasts were harvested from human donors and patients prepared. Microarray and gene-expression profiling, Chromatin immunoprecipitation microarray analysis, and Methylated DNA isolation assay-assisted CpG microarrays was performed in Wild-type and GCD2-homozygous human cells. RESULTS: Transcription and extracellular-secretion levels of TGFBIp were high in normal cells compared with those in GCD2-derived cells and were related to H3K4me3 levels but not to DNA methylation over the TGFBI locus. TGFβ1 increased the expression of TGFBIp and the ECM-associated genes connective tissue growth factor, collagen-α2[Ι], and plasminogen activator inhibitor-1 in normal corneal fibroblasts. Increased levels of gene-activating markers (H3K4me1/3) and decreased levels of repressive markers (H3K27me3) at the promoters of those gene accompanied the changes in expression. TGFβ1 also increased recruitment of the H3K4 methyltransferase MLL1 and of SET7/9 and also the binding of Smad3 to the promoters. Knockdown of both MLL1 and SET7/9 significantly blocked the TGFβ1-induced gene expression and inhibited TGFβ1-induced changes in promoter H3K4me1/3 levels. Those effects were very weak, however, in GCD2-derived corneal fibroblasts. CONCLUSIONS: Taken together, the results show the functional role of H3K4me in TGFβ1-mediated TGFBIp and ECM gene expression in corneal fibroblasts. Pharmacologic and other therapies that regulate these modifications could have potential cornea-protective effects for granular corneal dystrophy.ope

Inhibition of TGFBIp expression by lithium: implications for TGFBI-linked corneal dystrophy therapy

PURPOSE. The purpose of this study was to investigate the effects and molecular mechanisms of lithium on inhibition of TGFBIp expression as a potential therapy for TGFBI-linked corneal dystrophy. METHODS. Primary culture corneal fibroblasts were isolated from the corneas of healthy subjects and patients with granular corneal dystrophy type 2 (GCD2) with a homozygous mutation in TGFBI R124H. Levels of TGFBIp and its mRNA in corneal fibroblasts treated with various lithium (LiCl) concentrations were analyzed by Western blot, RT-PCR, and quantitative real-time PCR. RESULTS. LiCl treatment reduced the expression levels of normal and mutant TGFBIp in a dose- and a time-dependent manner. Furthermore, TGF-β1-induced TGFBIp expression decreased by 35% and 67% after treatment with 5 mM and 10 mM LiCl, respectively. LiCl decreased the level of pSmad3 (S423/425) in a dose-dependent manner. Furthermore, LiCl increased the level of pGSK-3α/β (S21/9) in a dose-dependent manner. Also observed was the interaction between GSK-3β and Smad3, which was enhanced by lithium. In addition, Western blot analysis showed that the ratio of LC3-II/LC3-I in corneal fibroblasts increased after LiCl treatment. Cell viability at different doses was greater than 98%, indicating that LiCl did not induce significant corneal fibroblast death. Finally, the observed attenuating effects of LiCl on TGFBIp expression were not the results of cell death. CONCLUSIONS. The accumulation of mutant TGFBIp ultimately leads to the histopathologic and clinical manifestations associated with TGFBI-linked corneal dystrophy. These data strongly suggest that lithium may be used for the prevention or treatment of this disease.ope

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

PURPOSE: The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. METHODS: Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. RESULTS: MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. CONCLUSIONS: MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.ope

Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis

Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.ope