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Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.

ABSTRACT: Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGF?)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGF?-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation.

Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGF?1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGF?-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (?-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis.

Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGF?1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in ?-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGF?1 showed the expected low expression of ?-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGF?-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGF?-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein.

Conclusions: Id3 regulates TGF?-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

SUBMITTER: Gupta S

PROVIDER: S-EPMC7700910 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Role of <i>inhibitor of differentiation 3 gene</i> in cellular differentiation of human corneal stromal fibroblasts.

Gupta Suneel S Martin Lynn M LM Sinha Nishant R NR Smith Kaitlin E KE Sinha Prashant R PR Dailey Emilee M EM Hesemann Nathan P NP Mohan Rajiv R RR

Molecular vision 20201125

<h4>Purpose</h4>Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The <i>inhibitor of differentiation 3 (Id3) gene</i>, a member of the <i>Id gene</i> family, governs the expression and progression of transforming growth factor beta (TGFβ)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal in ...[more]

PMID: 33273801

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Project description:Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-β (TGFβ). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with corneal fibrosis resulting in severe vision loss. The objective of this study was to determine the effect of hepatocyte growth factor (HGF), which can modulate TGFβ signaling, on corneal myofibroblast transformation by analyzing the expression of α-smooth muscle actin (αSMA) as a marker of myofibroblast phenotype particularly as it relates to biomechanical cues. Human corneal fibroblasts were cultured on tissue culture plastic (>1 GPa) or hydrogel substrates mimicking human normal or wounded corneal stiffness (25 and 75 kPa) in media containing TGFβ1 ± HGF. The expression of αSMA was analyzed by quantitative PCR, Western blot and immunocytochemistry. Cellular stiffness, which is correlated with cellular phenotype, was measured by atomic force microscopy (AFM). In primary human corneal fibroblasts, the mRNA expression of αSMA showed a clear dose response to TGFβ1. The expression was significantly suppressed when cells were incubated with 20 ng/ml HGF in the presence of 2 ng/ml of TGFβ1. The protein expression of αSMA induced by 5 ng/ml TGFβ1 was also decreased by 20 ng/ml of HGF. Cells cultured on hydrogels mimicking human normal (25 kPa) and fibrotic (75 kPa) cornea also showed an inhibitory effect of HGF on αSMA expression in the presence or absence of TGFβ1. Cellular stiffness was decreased by HGF in the presence of TGFβ1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGFβ1 in human corneal stromal cells. These data suggest that HGF holds the potential as a therapeutic agent to improve wound healing outcomes by minimizing corneal fibrosis.

Project description:BackgroundHuman corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth.MethodsThree placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry.ResultsAME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures.ConclusionAME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Project description:The need for an autologous cell source for bone tissue engineering and medical applications has led researchers to explore multipotent mesenchymal stromal cells (MSC), which show stem cell plasticity, in various human tissues. However, MSC with different tissue origins vary in their biological properties and their capability for osteogenic differentiation. Furthermore, MSC-based therapies require large-scale ex vivo expansion, accompanied by cell type-specific replicative senescence, which affects osteogenic differentiation. To elucidate cell type-specific differences in the osteogenic differentiation potential and replicative senescence, we analysed the impact of BMP and TGF-β signaling in adipose-derived stromal cells (ASC), fibroblasts (FB), and dental pulp stromal cells (DSC). We used inhibitors of BMP and TGF-β signaling, such as SB431542, dorsomorphin and/or a supplemental addition of BMP-2. The expression of high-affinity binding receptors for BMP-2 and calcium deposition with alizarin red S were evaluated to assess osteogenic differentiation potential. Our study demonstrated that TGF-β signaling inhibits osteogenic differentiation of ASC, DSC and FB in the early cell culture passages. Moreover, DSC had the best osteogenic differentiation potential and an activation of BMP signaling with BMP-2 could further enhance this capacity. This phenomenon is likely due to an increased expression of activin receptor-like kinase-3 and -6. However, in DSC with replicative senescence (in cell culture passage 10), osteogenic differentiation sharply decreased, and the simultaneous use of BMP-2 and SB431542 did not result in further improvement of this process. In comparison, ASC retain a similar osteogenic differentiation potential regardless of whether they were in the early (cell culture passage 3) or later (cell culture passage 10) stages. Our study elucidated that ASC, DSC, and FB vary functionally in their osteogenic differentiation, depending on their tissue origin and replicative senescence. Therefore, our study provides important insights for cell-based therapies to optimize prospective bone tissue engineering strategies.

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Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.

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Role of <i>inhibitor of differentiation 3 gene</i> in cellular differentiation of human corneal stromal fibroblasts.

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