Effects of adeno-associated virus (AAV) of transforming growth factors β_1 and β_3 (TGFβ_(1,3)) on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type II of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type II were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type II, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Effects of adeno-associated virus (AAV) of transforming growth factors β1 and β3 (TGFβ1,3) on promoting synthesis of glycosaminoglycan and collagen type II of de-differentiated nucleus pulposus (NP) cells

The effects of AAV-TGFβ1 and AAV-TGFβ3 on promoting synthesis of glycosaminoglycan and collagen type Ⅱ of dedifferentiated rabbit lumbar disc NP cells were studied in this work. The rabbit lumbar disc NP cells were isolated and cultured. The earlier and later dedifferentiated NP cells were established by subculture. The AAV transfection efficiency to dedifferentiated NP cells was analyzed with AAV-EGFP in vitro. After dedifferentiated NP cells were transfected by AAV-TGFβ1 or AAV-TGFβ3, their biological effects on promoting synthesis of glycosaminoglycan or collagen type Ⅱ were detected and compared by the methods of 35S incorporation or immunoblotting. The experimental results showed that AAV could transfect efficiently the earlier dedifferentiated NP cells, but its transfection rate was shown to be at a low level to the later dedifferentiated NP cells. Both AAV-TGFβ1 and AAV-TGFβ3 could promote the earlier dedifferentiated NP cells to synthesize glycosaminoglycan and collagen type Ⅱ, and the effect of AAV-TGFβ1 was better than that of AAV-TGFβ3. For the later dedifferentiated NP cells, the AAV-TGFβ3 could promote their synthesis, but AAV-TGFβ1 could slightly inhibit their synthesis. Therefore, AAV-TGFβ1 and AAV-TGFβ3 could be used for the earlier dedifferentiated NP cells, and the TGFβ3 could be used as the objective gene for the later dedifferentiated NP cells.     

Autophagy promotes intracellular degradation of type I collagen induced by transforming growth factor (TGF)-β1

Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A(1) also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-β1. We also show that TGF-β1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-β1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-β1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.     

Heterobicyclic inhibitors of transforming growth factor beta receptor I (TGFβRI)

The TGFβ-TGFβR signaling pathway has been reported to play a protective role in the later stages of tumorigenesis via increasing immunosuppressive Treg cells and facilitating the epithelial to mesenchymal transition (EMT). Therefore, inhibition of TGFβR has the potential to enhance antitumor immunity. Herein we disclose the identification and optimization of novel heterobicyclic inhibitors of TGFβRI that demonstrate potent inhibition of SMAD phosphorylation. Application of structure-based drug design to the novel pyrrolotriazine chemotype resulted in improved binding affinity (Ki apparent?=?0.14?nM), long residence time (T_1/2?>?120?min) and significantly improved potency in the PSMAD cellular assay (IC₅₀?=?24?nM). Several analogs inhibited phosphorylation of SMAD both in vitro and in vivo. Additionally, inhibition of TGFβ-stimulated phospho-SMAD was observed in primary human T cells.     

Combinatorial role of two G-quadruplexes in 5′ UTR of transforming growth factor β2 (TGFβ2)

Albeit most studies demonstrate the inhibitory role of G-quadruplex in the 5′ Untranslated Region (5′ UTR), our previous report depicted its completely contrasting activating role in the 5′ UTR of transforming growth factor β2 (TGFβ2) mRNA. Therefore, we screened the 5′ UTR of TGFβ2 manually and identified a second putative G-quadruplex sequence. Our in vitro experiments encompassing CD and UV spectroscopy confirmed the ability of this sequence to form a G-quadruplex and in cellulo studies further indicated its activating role in modulation of TGFβ2 gene expression. Our study suggests that these two 5′ UTR G-quadruplexes most probably operate additively to substantially increase gene expression of TGFβ2. Neither of the two G-quadruplex alone is sufficient enough to drastically augment protein production. Both G-quadruplexes are essential for increasing protein output. To the best of our knowledge, our study is the first report showcasing the combinatorial role of two G-quadruplexes in the 5′ UTR of an mRNA.     

TGFβ (transforming growth factor β) and keratocyte motility in 24 h zebrafish explant cultures

Fish keratocytes are used as a model system for the study of the mechanics of cell motility because of their characteristic rapid, smooth gliding motion, but little work has been done on the regulation of fish keratocyte movement. As TGFβ (transforming growth factor β) plays multiple roles in primary human keratinocyte cell migration, we investigated the possible involvement of TGFβ in fish keratocyte migration. Studying the involvement of TGFβ1 in 24 h keratocyte explant allows the examination of the cells before alterations in cellular physiology occur due to extended culture times. During this initial period, TGFβ levels increase 6.2‐fold in SFM (serum‐free medium) and 2.4‐fold in SFM+2% FBS (fetal bovine serum), while TGFβ1 and TGFβRII (TGFβ receptor II) mRNA levels increase ～3‐ and ～5‐fold respectively in each culture condition. Two measures of motility, cell sheet area and migration distance, vary with the amount of exogenous TGFβ1 and culture media. The addition of 100 ng/ml exogenous TGFβ1 in SFM increases both measures [3.3‐fold (P=4.5 × 10^?5) and 26% (P=2.1 × 10^?2) respectively]. In contrast, 100 ng/ml of exogenous TGFβ1 in medium containing 2% FBS decreases migration distance by 2.1‐fold (P=1.7 × 10^?7), but does not affect sheet area. TGFβ1 (10 ng/ml) has little effect on cell sheet area in SFM cultures, but leads to a 1.8‐fold increase (P=1.5 × 10^?2) with 2% FBS. The variable response to TGFβ1 may be, at least in part, explained by the effect of 2% FBS on cell morphology, mode of motility and expression of endogenous TGFβ1 and TGFβRII. Together, these results suggest that expression of TGFβ and its receptor are up‐regulated during zebrafish keratocyte explant culture and that TGFβ promotes fish keratocyte migration.     

Potential role of follicle-stimulating hormone (FSH) and transforming growth factor (TGFβ1) in the regulation of ovarian angiogenesis

Angiogenesis occurs during ovarian follicle development and luteinization. Pituitary secreted FSH was reported to stimulate the expression of endothelial mitogen VEGF in granulosa cells. And, intraovarian cytokine transforming growth factor (TGF)β1 is known to facilitate FSH‐induced differentiation of ovarian granulosa cells. This intrigues us to investigate the potential role of FSH and TGFβ1 regulation of granulosa cell function in relation to ovarian angiogenesis. Granulosa cells were isolated from gonadotropin‐primed immature rats and treated once with FSH and/or TGFβ1 for 48 h, and the angiogenic potential of conditioned media (granulosa cell culture conditioned media; GCCM) was determined using an in vitro assay with aortic ring embedded in collagen gel and immunoblotting. FSH and TGFβ1 increased the secreted angiogenic activity in granulosa cells (FSH + TGFβ1 > FSH ≈ TGFβ1 > control) that was partly attributed to the increased secretion of pro‐angiogenic factors VEGF and PDGF‐B. This is further supported by the evidence that pre‐treatment with inhibitor of VEGF receptor‐2 (Ki8751) or PDGF receptor (AG1296) throughout or only during the first 2‐day aortic ring culture period suppressed microvessel growth in GCCM‐treated groups, and also inhibited the FSH + TGFβ1‐GCCM‐stimulated release of matrix remodeling‐associated gelatinase activities. Interestingly, pre‐treatment of AG1296 at late stage suppressed GCCM‐induced microvessel growth and stability with demise of endothelial and mural cells. Together, we provide original findings that both FSH and TGFβ1 increased the secretion of VEGF and PDGF‐B, and that in turn up‐regulated the angiogenic activity in rat ovarian granulosa cells. This implicates that FSH and TGFβ1 play important roles in regulation of ovarian angiogenesis during follicle development. J. Cell. Physiol. 226: 1608–1619, 2011. © 2010 Wiley‐Liss, Inc.     

Effects of transforming growth factor-β on collagen gene expression and collagen synthesis level in mineralizing cultures of osteoblast-like cell line,MC3T3-E1

• 1.1. High levels of type I collagen mRNA and [³H]proline incorporation into collagenase digestable protein by MC3T3-E1 cells were detected during the first 7 days of culture, after which they declined.
• 2.2. Type I collagen gene expression was stimulated by TGF-β in the early culture stage when the collagen gene expression was fully functioning.
• 3.3. However, these stimulatory effects disappeared at the differentiation stages. Although collagen gene expression was stimulated by TGF-β (2.0 ng/ml) in early culture, collagen synthesis in medium was not.
• 4.4. This study shows that collagen synthesis and collagen gene expression were affected by the state of differentiation in MC3T3-E1 cells and that the rate of stimulation by TGF-β in collagen gene expression decreased over time in culture.

     

In situ measurement of collagen synthesis by human bone cells with a Sirius Red-based colorimetric microassay: effects of transforming growth factor β2 and ascorbic acid 2-phosphate

Staining of collagens by Sirius Red, a standard histological procedure, was applied to quantify collagen synthesis in human osteoblast-like cell cultures in situ. After morphological analysis of the deposited material, the stain was dissolved and its optical density determined spectrophotometrically using a microtiter plate assay system. The method was sensitive with a detection limit for collagen synthesized by 3000 normal human periosteal cells. The assay is easy to perform and specific with respect to different extracellular materials, for example, collagen types I and III were well stained, collagen type IV and laminin exhibited only low staining, and fibronectin, chondroitin sulfate, dermatan sulfate, and amyloid β were negative. A major advantage of the method is the combination of identification of collagen-producing cells in situ with subsequent spectrophotometric quantification of the dissolved stain. Thus it is possible to obtain information on cell morphology, active sites of collagen deposition in a cell culture, microscopic detection of high-and low-producer cells prior to dissolution and quantification of the deposited material. In this regard the assay is superior to either radioactive labeling, hydroxyproline determination, or Sirius Red-based colorimetric assays with cell lysates. Since the quantification is based on microtiter plate reading, the method can be recommended for the screening of large quantities of samples. Accepted: 30 June 1999     

Effects of growth factors FGF4, TGFα, and TGFβ1 on development of parthenogenetic embryos of C57BL/6 mice

We studied the effects of three growth factors, fibroblast growth factor (FGF4), transforming growth factor (TGF), and transforming growth factor 1 (TGF1), on development of diploid parthenogenetic embryos of C57BL/6 mice, which are not capable of developing to somatic stages. Parthenogenetic embryos were treated with growth factors at optimal doses in vitro at the morula-blastocyst stages and transplanted in the uterus of pseudopregnant females. FGF4 and TGF improved the development of parthenogenetic embryos at the preimplantation stages and the number of blastocysts increased under the influence of TGF. All three growth factors improved the implantation of embryos in the uterus. When FGF4 or TGF1 2.4 were added to the nutrient medium, 2.4 or 1.6%, respectively, of parthenogenetic embryos reached the somatic stages in utero. No somitic embryos were observed in the control. The treatment of parthenogenetic embryos with two growth factors, FGF4 and TGF1 , simultaneously increased the amount of somatic embryos to 7.5%, while combination of three growth factors in creased the amount of such embryos to 16.7%. In the latter case, some parthenogenetic embryos reached the stage of 25–27 pairs of somites and were 2.0–2.5 mm long. The data we obtained suggest that, when combined, the growth factors FGF4, TGF, and TGF1 possessed a synergistic effect leading to a significant improvement of the development of parthenogenetic C57BL/6 embryos.__________Translated from Ontogenez, Vol. 36, No. 2, 2005, pp. 145–150.Original Russian Text Copyright © 2005 by Penkov, Platonov, Dimitrov, Mironova, Konyukhov.     

Synergistic role of c-Myc and ERK1/2 in the mitogenic response to TGFβ-1 in cultured rat nucleus pulposus cells

Introduction  

Although transforming growth factor β1 (TGFβ1) is known to be a potent inhibitor of proliferation in most cell types, it accelerates proliferation in certain mesenchymal cells, such as articular chondrocytes and nucleus pulposus cells. The low ability for self-renewal of nucleus pulposus cells is one obstacle in developing new therapeutic options for intervertebral disc diseases, and utilizing cytokines is one of the strategies to regulate nucleus pulposus cell proliferation. However, the precise cell cycle progression and molecular mechanisms by which TGFβ1 stimulates cell growth remain unclear. The aim of this study was to elucidate a mechanism that enables cell proliferation with TGFβ1 stimulation.     

Oral glucosamine increases expression of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) mRNA in rat cartilage and kidney: implications for human efficacy and toxicity

Glucosamine is used for alleviating pain in osteoarthritis. Clinical trials have reported that glucosamine has equivocal efficacy. Glucosamine is also used in cell cultures to stimulate hexosamine flux and protein O-glycosylation, but at many-fold greater concentrations than those in human plasma following oral dosing. Lean Zucker rats were dosed orally for 6 weeks with glucosamine hydrochloride at doses (0–600 mg/kg/day) that produced peak serum concentrations of <1–35 μM, spanning the human exposure range. Relative expression of both TGFβ1 and CTGF mRNA were significantly increased up to 2.3-fold in liver, kidney and articular cartilage when evaluated 4 h after final dose. Apparent threshold serum glucosamine (C_max) concentration required to increase TGFβ1 expression in cartilage was 10–20 μM. These increases were associated with significant increases in UDP-N-acetylglucosamine concentrations suggesting increased hexosamine flux. Both TGFβ1 and CTGF are mediators of chondrocyte proliferation and cartilage repair. Study demonstrates that oral glucosamine doses that produce clinically relevant serum glucosamine concentrations can induce tissue TGFβ1 and CTGF expression in vivo and provides a mechanistic rationale for reported beneficial effects of glucosamine therapy. Induction of renal TGFβ1 and CTGF mRNA suggests that potential sclerotic side-effects may occur following consumption of potent glucosamine preparations.     

Paramylon (β-1,3-glucan) content in wild type and WZSL mutant of Euglena gracilis. Effects of growth conditions

The effects of growth conditions on paramylon (a -1,3-glucanreserve carbohydrate) content were examined in the photosyntheticwild-type and a spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. This carbohydrate is known toshow interesting applications in human and veterinary medicine, asimmunostimulant and immunopotentiator. For both strains, the synthesisof reserve depends mainly on the growth conditions, i.e. light or dark. Thehighest amount of paramylon is accumulated by the mutant (90% dryweight) under dark conditions and with glucose as the carbon source. Thesefindings are discussed in terms of feasibility of exploitation of both strainsas an alternative source of -glucan, and of the importance of thechloroplast compartment in the synthesis of this compound.     

Adamts5 deletion blocks murine dermal repair through CD44-mediated aggrecan accumulation and modulation of transforming growth factor β1 (TGFβ1) signaling

ADAMTS5 has been implicated in the degradation of cartilage aggrecan in human osteoarthritis. Here, we describe a novel role for the enzyme in the regulation of TGFβ1 signaling in dermal fibroblasts both in vivo and in vitro. Adamts5(-/-) mice, generated by deletion of exon 2, exhibit impaired contraction and dermal collagen deposition in an excisional wound healing model. This was accompanied by accumulation in the dermal layer of cell aggregates and fibroblastic cells surrounded by a pericellular matrix enriched in full-length aggrecan. Adamts5(-/-) wounds exhibit low expression (relative to wild type) of collagen type I and type III but show a persistently elevated expression of tgfbRII and alk1. Aggrecan deposition and impaired dermal repair in Adamts5(-/-) mice are both dependent on CD44, and Cd44(-/-)/Adamts5(-/-) mice display robust activation of TGFβ receptor II and collagen type III expression and the dermal regeneration seen in WT mice. TGFβ1 treatment of newborn fibroblasts from wild type mice results in Smad2/3 phosphorylation, whereas cells from Adamts5(-/-) mice phosphorylate Smad1/5/8. The altered TGFβ1 response in the Adamts5(-/-) cells is dependent on the presence of aggrecan and expression of CD44, because Cd44(-/-)/Adamts5(-/-) cells respond like WT cells. We propose that ADAMTS5 deficiency in fibrous tissues results in a poor repair response due to the accumulation of aggrecan in the pericellular matrix of fibroblast progenitor cells, which prevents their transition to mature fibroblasts. Thus, the capacity of ADAMTS5 to modulate critical tissue repair signaling events suggests a unique role for this enzyme, which sets it apart from other members of the ADAMTS family of proteases.     

Repression of β-actin synthesis and persistence of ribosomal protein synthesis after infection of HeLa cells by herpes simplex virus type 1 infection are under translational control

Synthesis and assembly of ribosomal proteins into mature ribosomes persist late after infection of cells with herpes simplex virus type 1, while synthesis of β-actin is drastically shut off. Since mRNAs encoding ribosomal proteins and β-actin undergo concomitant degradation in infected HeLa cells, we have advanced the hypothesis that translation of the remaining mRNAs is differentially controlled after infection. The behaviour of mRNAs for three ribosomal proteins and for β-actin was investigated during the course of infection. In uninfected cells, β-actin mRNAs are associated with large polyribosomes, while only a part of ribosomal protein mRNAs are present in polyribosomes. In the course of infection, β-actin mRNAs are released from the ribosomes and are sequestered with 40S ribosomal subunits. Simultaneously, ribosomal protein mRNAs become associated with an increased number of ribosomes, even late in infection. In addition, virally induced phosphorylation of ribosomal protein S6 is more efficient in pre-existing ribosomes than in newly assembled ribosomes. These results indicate that in infected cells (i) translation of β-actin mRNA is selectively inhibited at a step necessary for binding the 60S ribosomal subunits; (ii) the rate of initiation of translation of ribosomal protein mRNAs increases after infection; and (iii) it is likely that translation of ribosomal protein mRNAs takes place preferentially on pre-existing ribosomes. Received: 5 February 1997 / Accepted: 28 May 1997     

Effects of transforming growth factor β1 and basic fibroblast growth factor on lipoprotein lipase activity in primary cultures of chicken (Gallus domesticus) adipocyte precursors

• 1.1. The effect of TGF-β and bFGF on lipoprotein lipase activity in chicken adipocyte precursors was investigated.
• 2.2. Lipoprotein lipase activity was reduced by up to 80% by incubation with TGF-β whereas bFGF had no effect.
• 3.3. Contrary to that found with the 3T3-L1 preadipocyte cell line it was not necessary for TGF-β to be present prior to the start of differentiation in order to be effective.
• 4.4. Incubation of adipocyte precursors with actinomycin D abolished the effect of TGF-β suggesting that synthesis of a protein effector is required.
• 5.5. These results indicate differences in responsiveness to TGF-β and bFGF between primary chicken adipocyte precursors and some preadipocyte cell lines.

     

The response of foetal annulus fibrosus cells to growth factors: modulation of matrix synthesis by TGF-β1 and IGF-1

The annulus fibrosus of the intervertebral disc is a complex radial-ply tissue that derives initially from segmental condensations of axial mesenchyme surrounding the notochord. These mesenchymal condensations differentiate into the early annulus fibrosus during foetal development—their outer part becoming fibrous, containing collagen type I; and their inner part cartilaginous, containing type II collagen and aggrecan. With post-natal growth and ageing, there is a switch from type I to type II collagen and an increase in proteoglycan synthesis in the outer annulus. This fibrocartilaginous metaplasia appears to occur in response to compressive loading of the tissue as occurs in tendons that wrap around bony pulleys, and driven by growth factors, such as TGF-β. In this study, using high-density micromass cultures, we have assessed the response of foetal outer annulus cells to growth factor stimulation with TGF-β1 and IGF-1, growth factors known to occur within the developing disc. We qualitatively and quantitatively describe the stimulatory effects of these growth factors, both alone and in combination, on the synthesis of sulphated glycosaminoglycan, and collagen types I and II by annulus cells. We show a potential role for TGF-β1 in pushing cells towards a fibrocartilaginous phenotype, with possible complementary effects of IGF-1.     

Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A₁, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A₁ caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.     

Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A₁, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A₁ caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.