Proteoglycan Expression during Transforming Growth Factor β-induced Keratocyte-Myofibroblast Transdifferentiation

Funderburgh, James L.; Funderburgh, Martha L.; Mann, Mary M.; Corpuz, Lolita M.; Roth, Mary R.

doi:10.1074/jbc.m107596200

Cited by 153 publications

(152 citation statements)

References 39 publications

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“…The glycosaminoglycans also change in a manner similar to that seen in scar tissue (8). Secretion of sulfated keratan sulfate is reduced, and dermatan sulfate made by these cells is more highly sulfated and more abundant (10). HA is also up-regulated in this in vitro model of fibrosis (8).…”

Section: Hyaluronan (Ha)mentioning

confidence: 63%

“…On exposure to serum and TGF␤, keratocytes alter their morphology and matrix secretion in a manner similar to cells in healing stromal wounds (8,10). Expression of keratocan, a unique stromal keratan sulfate proteoglycan, is strongly down-regulated and that of biglycan, a dermatan sulfate proteoglycan not normally present in stroma, is increased markedly (10). The glycosaminoglycans also change in a manner similar to that seen in scar tissue (8).…”

Section: Hyaluronan (Ha)mentioning

confidence: 73%

“…In vitro, keratocytes under quiescent serum-free conditions secrete matrix components similar or identical to those they produce in vivo (7)(8)(9). On exposure to serum and TGF␤, keratocytes alter their morphology and matrix secretion in a manner similar to cells in healing stromal wounds (8,10). Expression of keratocan, a unique stromal keratan sulfate proteoglycan, is strongly down-regulated and that of biglycan, a dermatan sulfate proteoglycan not normally present in stroma, is increased markedly (10).…”

Section: Hyaluronan (Ha)mentioning

confidence: 89%

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A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor β

Guo

Kanter

Funderburgh

et al. 2007

Journal of Biological Chemistry

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Keratocytes of the corneal stroma produce transparent extracellular matrix devoid of hyaluronan (HA); however, in corneal pathologies and wounds, HA is abundant. We previously showed primary keratocytes cultured under serum-free conditions to secrete matrix similar to that of normal stroma, but serum and transforming growth factor ␤ (TGF␤) induced secretion of fibrotic matrix components, including HA. This study found HA secretion by primary bovine keratocytes to increase rapidly in response to TGF␤, reaching a maximum in 12 h and then decreasing to <5% of the maximum by 48 h. Cell-free biosynthesis of HA by cell extracts also exhibited a transient peak at 12 h after TGF␤ treatment. mRNA for hyaluronan synthase enzymes HAS1 and HAS2 increased >10-and >50-fold, respectively, in 4 -6 h, decreasing to near original levels after 24 -48 h. Small interfering RNA against HAS2 inhibited the transient increase of HAS2 mRNA and completely blocked HA induction, but small interfering RNA to HAS1 had no effect on HA secretion. HAS2 mRNA was induced by a variety of mitogens, and TGF␤ acted synergistically to induce HAS2 by as much as 150-fold. In addition to HA synthesis, treatment with TGF␤ induced degradation of fluorescein-HA added to culture medium. These results show HA secretion by keratocytes to be initiated by a rapid transient increase in the HAS2 mRNA pool. The very rapid induction of HA expression in keratocytes suggests a functional role of this molecule in the fibrotic response of keratocytes to wound healing. Hyaluronan (HA)2 is a high molecular weight, nonsulfated glycosaminoglycan abundant in most tissues, where it acts as a hydrating agent and an organizer of extracellular matrix scaffolding via specific interactions with matrix proteins containing hyaluronectin domains (for review see Ref. 1). The corneal stroma, unlike most vertebrate tissues, is virtually devoid of HA. During active corneal wound healing and in corneas with various chronic pathologies, however, hyaluronan becomes abundant in the corneal stroma (2, 3).The corneal stroma maintains transparency to light by virtue of the highly organized structure of its collagenous extracellular matrix. Collagen fibrils of the stroma exhibit highly regular parallel alignment and spacing. This spacing is controlled by collagen-associated small leucine-rich proteoglycans that form glycosaminoglycan cross-links between adjacent fibrils (4). Disruption of the fibril spacing is a major cause of loss of corneal transparency in scarring and stromal pathological conditions (5). In scars, interfibrillar glycosaminoglycan cross-links are altered or eliminated, and spaces without fibrils, known as "lakes," have been identified (5). The almost ubiquitous presence of HA in nontransparent corneas suggests a relationship between the large hydrodynamic volume occupied by HA molecules and the disruption of the stromal ultrastructure. Additionally, the recent recognition of the diverse bioactivity of HA (6) raises the potential that matrix production by stromal keratocytes may...

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Section: Hyaluronan (Ha)mentioning

confidence: 63%

Section: Hyaluronan (Ha)mentioning

confidence: 73%

Section: Hyaluronan (Ha)mentioning

confidence: 89%

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A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor β

Guo

Kanter

Funderburgh

et al. 2007

Journal of Biological Chemistry

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“…Primary Culture of Corneal Fibroblasts-Primary corneal fibroblasts were obtained from fresh bovine corneas by collagenase digestion modified from the methods described by Funderburgh et al (34). Briefly, the central portions of fresh bovine corneas were incubated at 37°C in 2.4 units of dispase II (Roche Applied Science)/ Dulbecco's modified Eagle's medium (DMEM; Invitrogen) solution containing antibiotics (penicillin, 50 g/ml; streptomycin, 50 g/ml; amphotericin B, 2.5 g/ml) at 37°C for 3 h to remove the corneal epithelium and endothelium.…”

Section: Methodsmentioning

confidence: 99%

Coenzyme Q10 Rescues Ethanol-induced Corneal Fibroblast Apoptosis through the Inhibition of Caspase-2 Activation

Chen

Liou

Chen

et al. 2013

Journal of Biological Chemistry

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“…The keratocyte hallmarks, dendritic morphology, keratocan expression and KS production, are rapidly lost, reduced or altered in serum culture. Fibroblastic cells, in contrast, adopt elongated cell bodies and secrete biglycan and type III collagen, matrix components that are absent from healthy stroma but present in opaque scar tissues [20,30,31]. In hydrogel constructs, RT-PCR and confocal imaging indicated the production of normal stromal matrix components, particularly when primary and P1 cells are encapsulated.…”

Section: Discussionmentioning

confidence: 99%

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels

et al. 2008

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The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase-polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment.

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Proteoglycan Expression during Transforming Growth Factor β-induced Keratocyte-Myofibroblast Transdifferentiation

Abstract: Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor ␤ (TGF-

Cited by 153 publications

References 39 publications

A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor β

A Rapid Transient Increase in Hyaluronan Synthase-2 mRNA Initiates Secretion of Hyaluronan by Corneal Keratocytes in Response to Transforming Growth Factor β

Coenzyme Q10 Rescues Ethanol-induced Corneal Fibroblast Apoptosis through the Inhibition of Caspase-2 Activation

Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels

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