Celeste B. Rich

Insulin-like Growth Factor-I Regulates Transcription of the Elastin Gene through a Putative Retinoblastoma Control Element A ROLE FOR Sp3 ACTING AS A REPRESSOR OF ELASTIN GENE TRANSCRIPTION

Previous studies have demonstrated that insulin-like growth factor-I (IGF-I) increases elastin gene transcription in aortic smooth muscle cells and that this up-regulation is accompanied by a loss of protein binding to the proximal promoter. Sp1 has been identified as one of the factors whose binding is lost, and in the present study we show that Sp3 binding is also abrogated by IGF-I, but in a selected manner. In functional analyses using Drosophila SL-2 cells, Sp1 expression can drive transcription from the elastin proximal promoter, while co-expression of Sp3 results in a repression of Sp1 activity. Footprint and gel shift analyses position the IGF-I responsive sequences to a putative retinoblastoma control element (RCE). Mutation of the putative RCE sequence as assessed by transient transfection of smooth muscle cells results in an increase in reporter activity equal in magnitude to that conferred by IGF-I on the wild type promoter. Together these results support the hypothesis that IGF-I-mediated increase in elastin transcription occurs via a mechanism of derepression involving the abrogation of a repressor that appears to be Sp3 binding to the RCE.

Lysyl Oxidase Oxidizes Cell Membrane Proteins and Enhances the Chemotactic Response of Vascular Smooth Muscle Cells

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of LOX activity by β-aminopropionitrile (BAPN) in cultured rat aortic smooth muscle cells (SMCs) reduced the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. The chemotactic activity of PDGF-BB was significantly enhanced in the presence of a non-chemotactic concentration of LOX. We considered the possibility that extracellular LOX may oxidize cell surface proteins, including the PDGF receptor-β (PDGFR-β), to affect PDGF-BB-induced chemotaxis. Plasma membranes purified from control SMC contained oxidized PDGFR-β. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to these cells restored the profile of oxidized proteins toward that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells containing oxidized PDGFR-β was diminished by ∼2-fold when compared with cells in which oxidation by LOX was prevented by BAPN. Phosphorylated members of the PDGFR-β-dependent signal transduction pathway, including PDGFR-β, SHP2, AKT1, and ERK1/ERK2 (p44/42 MAPK), turned over faster in BAPN-treated than in control SMCs. LOX knock-out mouse embryonic fibroblasts mirrored the effect obtained with SMCs treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-β.

Regulation by P2X7: Epithelial Migration and Stromal Organization in the Cornea

PURPOSE Previously, the authors demonstrated that BzATP, a P2X(7) receptor agonist, enhanced corneal epithelial migration in vitro. The goal here was to characterize the role of the P2X(7) receptor in the repair of in vivo corneal epithelial debridement wounds and in the structural organization of the corneal stroma. METHODS Epithelial debridement was performed on P2X(7) knockout (P2X(7)(-/-)) and wild-type (WT) mice, and eyes were harvested after 16 hours. Corneas were stained with Richardson vital stain, and the wound area was recorded. Corneas were fixed and prepared for light microscopic, immunohistochemical, and electron microscopic analysis. Cuprolinic blue staining was performed to analyze stromal proteoglycans (PGs). Real-time PCR was performed to examine the expression of stromal collagens. RESULTS P2X(7) was present in the WT corneal epithelium but was not detected in P2X(7)(-/-) mice. Pannexin-1, a protein demonstrated to interact with P2X(7), was absent from the wound edge in P2X(7)(-/-). This was associated with a trend toward delayed corneal reepithelialization. Stromal ultrastructure and collagen alignment were altered in P2X(7)(-/-), and collagen fibrils had smaller diameters with a larger interfibrillar distances. Expression of collagen alpha1(I) and alpha3(v) was reduced. There were 30% fewer sulfated PGs along fibrils in the P2X(7)(-/-) stroma. CONCLUSIONS In the absence of the P2X(7) receptor, the expression of proteins in the corneal epithelium was altered and wound healing was compromised. Loss of receptor resulted in morphologic changes in the stroma, including changes in alignment of collagen fibrils, decreased expression of collagen, and smaller fibrils with fewer PGs per fibril.

Inhibition of histone acetyltransferase by glycosaminoglycans.

Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin‐derived oligosaccharides (>8 sugars) and N‐desulfated or O‐desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive‐like inhibitor causing an ∼50‐fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an ∼50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild‐type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation. J. Cell. Biochem. 105: 108–120, 2008.

The P2Y2 receptor mediates the epithelial injury response and cell migration

Injury to epithelial cells results in the release of ATP and stimulation of purinergic receptors and is thought to alter cell migration and wound repair. Medium from the injured cells triggers Ca(2+) mobilization and phosphorylation of ERK, both of which are inhibited if the medium is pretreated with apyrase. To understand the wound repair mechanism that occurs with injury, our goal was to determine which purinergic receptor(s) was the critical player in the wound response. We hypothesize that the P2Y(2) receptor is the key player in the response of corneal epithelial cells to cell damage and subsequent repair events. Cells transfected with short interfering RNA to either P2Y(2) or P2Y(4) were stimulated either by injury or addition of UTP and imaged using fluo 3-AM to monitor changes in fluorescence. When cells with downregulated P2Y(2) receptors were injured or stimulated with UTP, the intensity of the Ca(2+) release was reduced significantly. However, when cells with downregulated P2Y(4) receptors were stimulated, only the UTP-induced Ca(2+) response was reduced significantly. In addition, downregulation of the P2Y(2) receptor inhibited wound closure compared with unstimulated cells or cells transfected with nontargeting sequence. This downregulation resulted also in an attenuation in phosphorylation of Src and ERK. Together, these data indicate that the P2Y(2) receptor plays a major biological role in the corneal injury response and repair mechanisms.

In vitro model suggests oxidative stress involved in keratoconus disease

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Basic Fibroblast Growth Factor Decreases Elastin Gene Transcription through an AP1/cAMP-response Element Hybrid Site in the Distal Promoter

Previous studies demonstrated that basic fibroblast growth factor (bFGF) decreases elastin gene transcription in pulmonary fibroblasts. In this study we pursue the identification of the element and the trans-acting factors responsible. Gel shift analyses show that bFGF increases protein binding to a sequence located at −564 to −558 base pairs (bp), which possesses homology to both AP1 and cAMP-response consensus elements yet displays a unique affinity for heterodimer binding. Site-directed mutation of the −564- to −558-bp sequence results in an increase in promoter activity and abrogates the effect of bFGF. Western blot analysis shows that bFGF induces a sustained increase in the steady-state levels of Fra 1, and co-transfection of a Fra 1 expression vector with an elastin promoter reporter construct results in an inhibition of elastin promoter activity. Overall the results suggest that bFGF represses elastin gene transcription by increasing the amount of the Fra 1 that subsequently binds to the −564- to −558-bp as a heterodimer with c-Jun to form an inhibitory complex. We propose that the identified bFGF response element can serve to down-regulate elastin transcription in elastogenic cells and, conversely, can serve to up-regulate elastogenesis in cells where endogenous bFGF signaling is attenuated or altered.

Heparan sulfate depletion within pulmonary fibroblasts: Implications for elastogenesis and repair

We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan‐depleted fibroblasts were treated with porcine pancreatic elastase at 2–4‐day intervals and the elastase‐mediated release of fibroblast growth factor 2 (FGF‐2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF‐2 and glycosaminoglycans (GAG) from PG‐depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF‐2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.

Basic fibroblast growth factor decreases elastin gene transcription in aortic smooth muscle cells

The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run‐on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild‐type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein‐1‐cAMP response element (AP1/CRE) (−564 to −558‐bp) within the elastin promoter mediates the bFGF‐dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal‐regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra‐1. The addition of PD‐98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF‐dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury. J. Cell. Biochem. 85: 592–600, 2002.

Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death.