Yoshikuni Nakamura

Enhancement by neutrophils of collagen degradation by corneal fibroblasts

Activated corneal fbroblasts and infiltrated leukocytes are thought to contribute to corneal ulceration. The potential roles of neutrophil‐fibroblast and cell‐matrix interactions in the degradation of stromal collagen associated with corneal ulceration have now been investigated with the use of three‐dimensional cultures of rabbit cells in collagen gels. Degradation of collagen fibrils during culture was measured by spectrophotometric determination of released hydroxyproline. Whereas corneal fibroblasts alone degraded collagen fibrils to a small extent, neutrophils did not. However, the addition of neutrophils or neutrophil–conditioned medium (CM) to cultures of corneal fibroblasts resulted in a marked increase in the amount of collagen degraded by the fibroblasts. The effect of CM from neutrophils cultured in collagen gels on collagen degradation by corneal fibroblasts was greater than that of medium conditioned by neutrophils in monolayer culture. Immunoblot as well as reverse transcription and real‐time polymerase chain reaction analyses revealed that neutrophil–CM stimulated the synthesis of matrix metalloproteinase (MMP)‐1 and MMP‐3 by corneal fibroblasts. The stimulatory effect of neutrophils on collagen degradation by corneal fibroblasts was inhibited by the synthetic MMP inhibitor ilomastat and by interleukin‐1 (IL‐1) receptor antagonist. These results suggest that factors secreted by collagen‐stimulated neutrophils augment collagen degradation by corneal fibroblasts through a stimulatory effect on MMP synthesis and that IL‐1 released by neutrophils may contribute to this effect.

Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats

Purposes. 1) To measure the amount of calpain inhibitor SJA6017 taken up by lenses of young rats after administration; and 2) To test efficacy of SJA6017 against selenite cataract in regard to amelioration of proteolysis of lens protein and prevention of lens nuclear opacity. Methods. Selenite nuclear cataracts were produced by subcutaneous injection of an overdose of sodium selenite to 16-day-old rats. SJA6017 was administered daily using intraperitoneal injections at 100 mg/kg body weight/day for 4 days. Lenses were observed and photographed by slit lamp biomicroscopy, and scored into one of three stages. Enucleated lenses were also scored into one of four stages and lens opacities in the nuclear region were quantified by image analysis. Proteolysis of crystallins was detected by SDS-PAGE. The amount of SJA6017 taken up by the lens was detected with a column switching HPLC system. Results. Nuclear cataracts were visible in 31% of the animals receiving only selenite, while the frequency of nuclear cataract in the Se+SJA6017 group was reduced to only 16%. This effect of SJA6017 was confirmed by densitometric analysis as a reduction in the density of the nucleus. Similar proteolytic changes of crystallins occurred at all stages of selenite cataract formation. The amount of SJA6017 in the lens was detected at the level of 0.03 µM. Conclusions. Systemic SJA6017 was taken up by the lens, and SJA6017 ameliorated in vivo selenite cataract formation. These studies are important because they partially validate the biochemical rationale for developing non-surgical, drug treatments for cataract prevention in man.

Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins

The purpose of the present study was to compare the susceptibility of crystallins proteolyzed by ubiquitous calpain 2 and by lens-specific calpain Lp82 to insolubilization. To test this, transgenic (TG) mice expressing a calpain 2, in which the active site cysteine 105 was mutated to alanine, were produced. Expression of mutated calpain 2 was driven in lens by coupling the mutated gene to the betaB1-crystallin promoter. Light scattering was measured in solutions of lens proteins after activation of endogenous calpain 2 and/or Lp82. Mass spectrometric analysis was performed to determine the cleavage sites and the calpain responsible for insolubilization of crystallins. Lens proteins from TG mice incubated in vitro with calcium showed higher light scattering compared to proteins from wild type (WT) mice. alphaA-crystallin from TG mice was proteolyzed by Lp82. In contrast, alphaA-crystallin in lenses from WT mice were proteolyzed by both calpain 2 and Lp82. These results suggested that Lp82-induced proteolysis of crystallins caused increased susceptibility of truncated crystallins to in vitro precipitation. Since Lp82 is highest in young animals, Lp82-induced proteolysis and precipitation may be one of the factors responsible for the cataract formation in young rodents.

Integrin-mediated inhibition of interleukin-8 secretion from human neutrophils by collagen type I

The function of neutrophils in the inflammatory response is modulated by contact with ECM proteins. We have now investigated the effect of collagen type I on secretion of the cytokine IL‐8 by human neutrophils in vitro. Collagen type I inhibited the secretion of IL‐8 from neutrophils maintained under basal conditions or stimulated with fMLF. This effect was accompanied by down‐regulation of IL‐8 mRNA, and it appeared to be specific to collagen type I among ECM proteins, in that it was not observed with fibronectin or laminin. The inhibitory effect of collagen type I on IL‐8 secretion was dependent on collagen concentration and cell density. It was also abolished in the presence of antibodies to integrin α2β1 but was not affected by antibodies to integrin α5β1 or β4. Our results thus suggest that collagen type I inhibits the secretion of IL‐8 by human neutrophils in a selective manner and that this effect is mediated by the interaction of collagen with integrin α2β1.

Inhibition by all-trans retinoic acid of collagen degradation mediated by corneal fibroblasts

We examined the effect of all‐trans retinoic acid on collagen degradation mediated by corneal fibroblasts.

Coordinated Regulation of Palladin and α–Smooth Muscle Actin by Transforming Growth Factor–β in Human Corneal Fibroblasts

PURPOSE To investigate the role of palladin in the cornea, we examined expression of this actin assembly-related protein in normal, diseased, or injured corneal tissue as well as in cultured corneal fibroblasts. METHODS Expression of palladin and α-smooth muscle actin (α-SMA) in the rat cornea with an incision wound, in the normal and diseased human cornea, and in cultured human corneal fibroblasts was examined by immunofluorescence or immunoblot analysis. RESULTS The expression of both palladin and α-SMA was detected at the lesion site during wound healing in the rat cornea. Whereas neither palladin nor α-SMA was detected in the normal human cornea, the colocalization of both proteins was detected in diseased human corneas with underlying conditions characterized by the presence of fibrosis. The expression of both palladin and α-SMA in cultured human corneal fibroblasts was increased by transforming growth factor-β (TGF-β) in a manner sensitive to inhibition by blockers of Smad or mitogen-activated protein kinase (MAPK) signaling. Finally, RNA interference-mediated depletion of palladin attenuated the TGF-β-induced upregulation of α-SMA expression in human corneal fibroblasts as well as TGF-β-induced collagen gel contraction mediated by these cells. CONCLUSIONS Palladin is expressed in the rat and human cornea in association with scar formation. Expression of palladin in human corneal fibroblasts is increased by TGF-β in a manner dependent on Smad and MAPK signaling and is required for the TGF-β-induced upregulation of α-SMA.

Calpain-induced light scattering in young rat lenses is enhanced by UV-B

The purpose of this study is to determine if UV-B enhances light scattering after proteolysis of crystallins by calpains, and to determine if lens-specific calpain Lp82 is involved, along with m-calpain, in the mechanism of in vitro precipitation. Lens soluble proteins from young rats were hydrolyzed for 24 hr by endogenous lens calpains, and the proteins were further incubated for up to 7 days with periodic irradiation by UV-B. Light scattering was measured daily at 405 nm. SDS-PAGE and immunoblotting assessed proteolysis of crystallins, activation of calpains, and formation of high molecular weight aggregations. Appreciable light scattering occurred in lens soluble proteins after proteolysis of crystallins by m-calpain and Lp82. UV-B markedly enhanced this light scattering and the formation of higher molecular weight aggregates consisting of proteolyzed alpha- and beta- and intact gamma-crystallins. Calpain inhibitor E64 and antioxidants DTE or GSH prevented the light scattering. These results show that calpain-induced light scattering is enhanced by the natural oxidant UV-B. Activation of Lp82, along with m-calpain, contributed to the light scattering. The linkage between proteolysis and oxidation is important because both oxidation and truncation of crystallins are found in aged human lenses, which are constantly exposed to UV irradiation.