Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells

PURPOSE: This study investigated the regulated expression of collagenases (MMP-1, -8, and -13) and stromelysins (MMP-3, -10, and -11) by human corneal epithelial cells treated with IL-1 beta, TNF-alpha, and doxycycline, a medication used to treat ocular surface diseases. METHODS: Primary human corneal epithelial cell cultures were treated with IL-1 beta or TNF-alpha, with or without their corresponding inhibitors. Total RNA extracted from cells treated for 4 to 24 hours was subjected to semiquantitative RT-PCR and Northern hybridization. Conditioned media from 24-hour-treated cultures were evaluated for MMP production by ELISA and activity assays. RESULTS: Semiquantitative RT-PCR and Northern hybridization revealed that the mRNAs of MMP-1, -13, -3, -10, and -11 were dose dependently upregulated by IL-1 beta and TNF-alpha, whereas MMP-8 and -14 and tissue inhibitor of metalloproteinase (TIMP)-1 were not altered, in corneal epithelial cells. MMP ELISA and activity assays confirmed this dose-dependent increase in MMP-1, -13, -3, and -10 protein production in conditioned media by IL-1 beta and TNF-alpha. This stimulated production was inhibited by their neutralizing antibodies and by IL-1 receptor antagonist. Doxycycline suppressed stimulated MMP-1, -10, and -13 production at both the mRNA and protein levels. CONCLUSIONS: This study demonstrated that IL-1 beta and TNF-alpha upregulate collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, and -11) in human corneal epithelial cells. Doxycycline suppresses stimulated MMP-1, -13, and -10 at the mRNA and protein levels, which suggests that collagenases and stromelysins may play a role in the pathogenesis of sterile corneal ulceration and other ocular surface diseases.     

Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant

PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.     

Synthesis of type II interleukin-1 receptors by human corneal epithelial cells but not by keratocytes

PURPOSE. The purpose of this study was to determine whether human corneal epithelial cells and keratocytes synthesize both the soluble and membrane forms of the type II IL-1 receptor (IL-1RII). METHODS. Primary cell cultures of human corneal epithelial cells and keratocytes were established from human corneas. RT-PCR was used to analyze cell cultures for expression of IL-1RII mRNA. The capacity of corneal cells to synthesize membrane-bound IL-1RII was determined by immunofluorescence microscopy, whereas ELISA was used to quantitate synthesis of soluble IL-1RII after IL-1alpha and TNF-alpha stimulation. RESULTS. Corneal epithelial cells expressed IL-1RII mRNA. The cells also stained positive for membrane-bound IL-1RII, and media harvested from epithelial cell cultures contained up to 50 pg/ml of soluble IL-1RII. Both IL-1alpha and TNF-alpha significantly enhanced the amounts of soluble IL-1RII released from epithelial cell surfaces. In contrast to epithelial cells, corneal keratocytes did not express IL-1RII mRNA. Membrane-bound IL-1RII was not detected on keratocytes, nor was soluble IL-1RII detected in culture media harvested from these cells. CONCLUSIONS. Human corneal epithelial cells but not corneal keratocytes synthesize both membrane and soluble forms of IL-1RII. Because both forms of IL-1RII can function as IL-1alpha antagonists, the results suggest that human corneal epithelial cells but not corneal keratocytes have evolved the capacity to dampen IL-1alpha responses through the production of IL-1RII.     

Estrogen stimulation of proinflammatory cytokine and matrix metalloproteinase gene expression in human corneal epithelial cells

PURPOSE: Researchers have proposed that the secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs) by corneal epithelial cells contributes to the development of ocular surface inflammation in dry eye syndromes. We hypothesize that this cytokine and MMP production is promoted by estrogens, given that dry eye syndromes occur predominantly in women and estrogen therapy is associated with an increase in dry eye signs and symptoms. Moreover, corneal epithelial cells contain estrogen receptors and estrogens are known to influence other aspects of corneal immunity. The purpose of this study was to test our hypothesis. METHODS: Immortalized human corneal epithelial cells were exposed to vehicle, lipopolysaccharide (LPS), or varying concentrations of 17beta-estradiol. After 6 or 24 hours of hormone treatment, cells were harvested and processed for total RNA isolation, cDNA synthesis, and the analysis of cytokine and MMP mRNA levels by multiplex and real-time PCR methods. RESULTS: Our results demonstrate that 17beta-estradiol upregulates the expression of proinflammatory cytokine and MMP genes in human corneal epithelial cells. Estrogen administration significantly increased the levels of IL-1beta, IL-6, IL-8, and GM-CSF mRNAs, as well as MMP 2, 7, and 9 mRNAs, compared with those of placebo-treated controls. This estrogen effect was found after 6 and/or 24 hours of hormone treatment. An analogous stimulation of gene expression was observed following cellular exposure to LPS. CONCLUSIONS: Our findings show that 17beta-estradiol increases the expression of inflammatory genes in human corneal epithelial cells. This hormone action may play an etiologic role in the ocular surface inflammation of dry eye.     

IL-4 regulates chemokine production induced by TNF-α in keratocytes and corneal epithelial cells

BACKGROUND: Eosinophils are thought to play a major role in the pathogenesis of corneal lesions in ocular allergies. The regulation of chemokine production in corneal cells by the Th2 cytokine, interleukin 4 (IL-4), was examined in order to investigate its role in ocular allergies. METHODS: Pure cultures of human corneal epithelial cells and keratocytes were exposed to tumour necrosis factor alpha (TNF-alpha) and/or IL-4. 24 hours after exposure, culture supernatants were removed and concentrations of IL-8 and RANTES were quantified by ELISA assay. RESULTS: Simultaneous addition of IL-4 inhibited TNF-alpha induced IL-8 production in both corneal epithelial cells and keratocytes. TNF-alpha and IL-4 synergistically stimulated the production of RANTES in keratocytes. CONCLUSION: Differential regulation of chemokine production from corneal cells by IL-4 may play a role in the selective recruitment of predominantly eosinophils to the ocular surface in ocular allergies.     

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.     

Effect of metalloprotease inhibitors on corneal allograft survival

PURPOSE: The expression of Fas ligand (FasL) in the cornea is essential for corneal allograft acceptance in mice. Because the expression of FasL on the surface of cells is sensitive to cleavage with matrix metalloproteases (MMPs), this study examined whether inhibitors of MMPs would lead to increased FasL expression and improved corneal allograft survival. METHODS: Corneal endothelia derived from mice and humans were treated with MMP inhibitors, and FasL expression was examined. BALB/c mice were engrafted with C57BL/6 or C57BL/6-gld corneas and treated with an ointment containing the MMP inhibitor, doxycycline. Corneal allograft survival was monitored for 50 days. RESULTS: Corneal endothelial cells from both mice and humans displayed increased surface expression of FasL after treatment with MMP inhibitors. The increase in surface expression was further evidenced by the ability of these cells to kill Fas-expressing target cells. Mice treated with doxycycline after corneal allograft transplantation showed significantly prolonged allograft survival and an increase in the overall acceptance rate. CONCLUSIONS: MMP inhibitor treatment of cornea-derived endothelial cells results in increased FasL expression and function. MMP inhibitor treatment prolongs corneal allograft survival and results in a modest increase in corneal allograft acceptance.     

Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea.

PURPOSE: To determine the effect of interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha on cytokine, chemokine, and receptor expression in corneal stromal cells; the effect of corneal scrape injury on monocyte chemotactic and activating factor (MCAF) expression and monocyte-macrophage influx into the stroma; and the effect of MCAF and granulocyte colony-stimulating factor (G-CSF) microinjection on inflammatory cell infiltration into the stroma. METHODS: Gene array technology was used to evaluate changes in cytokine, chemokine, and receptor gene expression in stromal fibroblasts in response to IL-1alpha and TNFalpha. Expression of MCAF mRNA and protein was monitored with an RNase protection assay and Western blot analysis, respectively. Keratocyte MCAF protein expression in the rabbit cornea was detected with immunocytochemistry. After epithelial scrape injury, monocytes-macrophages were detected in rabbit corneas, by immunocytochemistry for monocyte-macrophage antigen. Inflammatory cell infiltration after MCAF and G-CSF microinjection into the stroma of mouse corneas was monitored with hematoxylin and eosin staining. RESULTS: IL-1alpha or TNFalpha upregulated the expression of several proinflammatory chemokines in stromal fibroblasts in culture. These included G-CSF, MCAF, neutrophil-activating peptide (ENA-78), and monocyte-derived neutrophil chemotactic factor (MDNCF). MCAF mRNA upregulation was confirmed by RNase protection assay, and MCAF protein was detected by Western blot analysis. MCAF protein was detected in keratocytes at 4 hours and 24 hours after epithelial injury, but not in keratocytes in the unwounded cornea. Corneal epithelial injury triggered the influx of monocytes-macrophages into the corneal stroma in the rabbit. Microinjection of MCAF and G-CSF into mouse cornea resulted in the influx of monocytes-macrophages and granulocytes, respectively, into the stroma. CONCLUSIONS: Proinflammatory chemokine induction in keratocytes is mediated by IL-1alpha and TNFalpha. The proinflammatory chemokines produced by the keratocytes probably trigger the influx of inflammatory cells into the stroma after epithelial injury associated with corneal surgery, contact lenses, or trauma.     

Altered expression of matrix metalloproteinases and their tissue inhibitors as possible contributors to corneal droplet formation in climatic droplet keratopathy

Purpose: Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP‐2, MMP‐9, MMP‐8 and MMP‐13 and their tissue inhibitors TIMP‐1 and TIMP‐2 in tear fluid of patients with CDK. Methods: Seventeen CDK patients and 10 controls living in Argentine Patagonia received a complete eye examination, and MMPs and TIMP‐1/2 were determined by immunofluorometric assay (IFMA), gelatin zymography and quantitative Western immunoblot analysis in tear samples. Results: The MMPs were detected mostly in their latent forms. The levels of MMP‐9 and MMP‐2 were found to be significantly elevated in CDK patients, whereas latent and active MMP‐8 levels were significantly enhanced in controls. There was no significant difference in the level of MMP‐13. TIMPs were found as part of complexes, and the TIMP‐1 levels were significantly lower in patients than controls. Conclusion: Elevated MMP‐2 and MMP‐9 levels have been implicated in the failure of corneal re‐epithelialization, and enhanced MMP‐2 and MMP‐9 levels in CDK patients suggest that these MMPs may play a role in corneal scarring in CDK. Elevated levels of MMP‐8 suggest a defensive role for this MMP in inflammatory reactions associated with recurring corneal traumas. Decreased expression of TIMP‐1 in CDK patients suggest deficient antiproteolytic shield likely to render the corneas of CDK patients vulnerable to enhanced MMPs. Overall, these data suggest a mechanistic link between MMPs and TIMP‐1 level in cornea and tears with corneal scarring in CDK.     

Production of IL-6 and IL-1 alpha by human corneal epithelial cells

By using enzyme-linked immunosorbent assay (ELISA), it was demonstrated that cultured human corneal epithelial cells produced interleukin-6 (IL-6) without any stimulation. As lipopolysaccharide (LPS)-stimulation did not induce further production of IL-6 and IL-1 alpha, it was suggested that IL-6 was produced naturally as in the case of IL-1 alpha production which has been previously reported by the author. Production of IL-1 alpha and IL-6 in the human corneal epithelial cells may play a role in amplifying immune responses on the ocular surface.     

Human corneal epithelial, stromal and endothelial cells produce interleukin-6]

We have been investigating the production of cytokines in ocular tissues. In this paper, we demonstrated the in vitro production of interleukin-6 (IL-6) by human corneal epithelial, stromal and endothelial cells using enzyme-linked immunosorbent assay (ELISA). In culture supernatant of the stromal cells, the production of immunoreactive IL-6 was induced, depending upon the doses of lipopolysaccharide (LPS) or IL-1 alpha added into the culture. Detectable IL-6 activity in the supernatant of the stromal cells was found 2 hours after addition of IL-1 alpha and the activity increased to a peak level at 48 hours. On the other hand, in the supernatant of the endothelial cells, IL-6 activity was found even in unstimulated-culture, and induced further by LPS stimulation. The molecular weights (MWs) of the IL-6 produced by the epithelial, stromal and endothelial cells were calculated by gel filtration as about 30 kDa. From Western blotting analysis, the MW of IL-6 produced by the stromal cells was also determined to be 30 kDa.     

多西环素对培养的人结膜上皮细胞炎性反应及凋亡的影响

目的探讨多西环素对体外培养的人结膜上皮细胞细胞黏附分子1（ICAM-1,CD54）、人类白细胞抗原DR（HLA—DR）、白细胞介素1β（IL-1β）表达及凋亡的影响。方法混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入γ干扰素（IFN-γ）0U／ml、IFN-γ300U／ml、IFN-γ300U／ml＋多西环素10μg／ml、IFN-γ300U／ml＋多西环素20μg／ml、IFN-γ300U／ml＋多西环素40μg／ml、IFN-γ300U／ml＋地塞米松100μg／ml,24h后收集细胞,流式细胞学检测CD54、HLA—DR及IL-1β的表达,Western blot检测CD54的表达。72h后收集细胞,碘化丙啶（PI）染色,流式细胞学检测凋亡。结果培养的正常人结膜上皮细胞可以表达少许CD54和IL-1β,加入IFN-γ后表达明显增加,加入多西环素和地塞米松后CD54和IL-1β的表达下降（P〈0．01）。随着所加多西环素浓度的增加,CD54和IL-1β的表达逐渐降低（P〈0．01）。培养的正常人结膜上皮细胞未发现明显的HLA—DR的表达及凋亡的产生,加入IFN-γ及多西环素后HLA—DR的表达及凋亡水平无明显变化（P〉0．05）。结论多西环素可抑制培养的正常人结膜上皮细胞CD54和IL-1β等炎性相关因子的产生,提示多西环素对干眼等非感染性眼表炎性疾病的治疗可能具有一定的应用前景。（中华眼科杂志,2005,41：842-846）     

TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of ocular surface diseases. This study investigated the regulated expression of gelatinases (MMP-2 and -9), collagenases (MMP-1 and -13) and stromelysins (MMP-3, -10, and -11) by TGF-beta1 in cultured human corneal epithelial cells. Primary human corneal epithelial cell cultures were grown to confluence and treated with different concentrations (0.1, 1.0, 10 ng ml(-1)) of TGF-beta1 in serum-free medium for 6-24 hr. Total RNA was isolated from cultured cells with or without TGF-beta1 treatment for 6 hr and subjected to semi-quantitative RT-PCR and Northern hybridization. Conditioned media were collected from cultures with or without TGF-beta1 treatment for 24 hr to evaluate the MMP production by ELISA and activity assays. Semi-quantitative RT-PCR revealed that the expressions of MMP-9, -1, -13, -3, -10 and -11 mRNA were up-regulated by TGF-beta1 in a concentration-dependent fashion, while MMP-2 and MMP-14 production did not change. Northern hybridization confirmed these findings. Gelatin zymography, MMP ELISA and activity assays showed concentration-dependent stimulated production and activity of MMP-9, -1, -13, -3 and -10 protein in the conditioned media of cultures treated for 24 hr with TGF-beta1. In conclusion, our results demonstrate that TGF-beta1 stimulates the expression and production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) in human corneal epithelial cells. These findings suggest that TGF-beta1 may play a role in the pathogenesis of MMP mediated ocular surface diseases, such as sterile corneal ulceration.     

Protective effect of uridine on cornea in a rabbit dry eye model

PURPOSE: To investigate the effect of uridine on cultured human corneal epithelial cells and keratocytes in vitro and to evaluate whether the application of uridine-containing eye drops could improve ocular surface health in an in vivo dry eye model. METHODS: Uridine was added to cultured epithelial cells (3 x 10(4) cells/well) and keratocytes (1 x 10(4) cells/well) at various concentrations (0.5-50 microM). Cytotoxicity was tested with the use of MTT assay, and the cells were assessed for apoptosis with the use of flow cytometry. Expressions of hyaluronic acid (HA), glycosaminoglycan (GAG), nitric oxide (NO), and matrix metalloproteinase (MMP)-9 were measured. In vivo, the degree of reepithelialization was assessed after topical application of uridine (100 microM) in a rabbit corneal wound model. Changes in tear production and conjunctival goblet cell counts were investigated after instillation of various concentrations of uridine-containing eye drops in a rabbit dry eye model. RESULTS: In vitro, uridine showed no cellular toxicity. It increased the biosynthesis of HA and GAG and reduced MMP-9 levels in cultured corneal epithelial cells and keratocytes. In vivo, uridine enhanced corneal wound healing and significantly increased the number of conjunctival goblet cells in rabbits. CONCLUSIONS: Uridine can restore the health of the ocular surface in a rabbit corneal wound and dry eye model.     

Stimulation of matrix metalloproteinases by hyperosmolarity via a JNK pathway in human corneal epithelial cells

PURPOSE: To investigate whether exposure of human corneal epithelial cells to hyperosmotic stress activates the c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway, and stimulates production of the matrix metalloproteinases (MMPs): gelatinase (MMP-9), collagenases (MMP-1 and -13), and stromelysin (MMP-3). METHODS: Primary human corneal epithelial cells cultured in normal osmolar medium (312 mOsM) were exposed to media with higher osmolarity (350-500 mOsM) achieved by adding NaCl, with or without SB202190, an inhibitor of the JNK pathway; dexamethasone; or doxycycline for different lengths of time. The conditioned media were collected after 24 hours of exposure for zymography and ELISA. Total RNA was extracted from cultures treated for 6 hours and subjected to semiquantitative RT-PCR. Cells treated for 5 to 60 minutes were lysed in RIPA buffer and subjected to Western blot with phospho (p)-specific antibodies against p-JNK and p-c-Jun. JNK1 activation was also detected with an immunoassay system. RESULTS: The concentrations of MMP-9, -1, and -3 proteins in 24-hour conditioned media of corneal epithelial cells progressively increased as the media's osmolarity was increased from 312 to 500 mOsM by the addition of NaCl. The concentration of MMP-13 progressively increased to a peak at 450 mOsM. Active p-JNK-1, p-JNK-2, and p-c-Jun were detected by Western blot as early as 5 minutes and peaked at 60 minutes in cells exposed to hyperosmolar media. The levels of p-JNK-1, p-JNK-2, and p-c-Jun correlated positively with the osmolarity of the culture media. The p-JNK inhibitor SB202190 and doxycycline markedly inhibited the stimulation of p-JNK-1, p-JNK-2, and p-c-Jun, as well as MMP-9, -1, -13, and -3 at both the mRNA and protein levels in the cells exposed to hyperosmolar media. CONCLUSIONS: Expression and production of MMP-9, -1, -13, and -3 by human corneal epithelial cells correlated positively with increasing media osmolarity. This increase was mediated at least in part through activation of the JNK SAPK pathway. Doxycycline, an agent used to treat MMP-mediated ocular surface disease, inhibited the hyperosmolarity-induced MMP production and JNK activation. The relevance of these findings to stimulated production of MMPs by the elevated tear osmolarity in dry eye remains to be determined.     

Comparative effects of estrogen on matrix metalloproteinases and cytokines in immortalized and primary human corneal epithelial cell cultures

PURPOSE: Recently, we found that 17beta-estradiol induces cytokine and MMP gene expression in SV40 immortalized human corneal epithelial cells (SV40 HCEs). The purpose of this study was to determine if 17beta-estradiol stimulates MMP secretion by cultures of SV40 HCEs and primary human corneal epithelial cells. Further, we determined if estrogen influenced cytokine and MMP gene expression in the primary cultures. METHODS: Gelatin zymography was used to identify MMP-2 and MMP-9 gelatinase activity in the culture medium from SV40 HCEs and primary human corneal epithelial cell cultures exposed to vehicle or 17beta-estradiol for 6 or 24 hour. In addition, 17beta-estradiol effects on cytokine and MMP gene expression in primary cultures were evaluated by real-time PCR methods. RESULTS: 17beta-estradiol had no significant effects on MMP activity in the culture media of either SV40 HCEs or primary corneal epithelial cell cultures. SV40 HCEs secreted predominantly MMP-2, whereas the primary cultures secreted predominantly MMP-9. In primary cultures, 17beta-estradiol caused a significant decrease of IL-6 and IL-8 gene expression, but had no effect on the levels of IL-1beta, MMP-2, and MMP-9 gene expression. CONCLUSIONS: Our findings suggest that 17beta-estradiol effects on gelatinase gene expression in SV40 HCEs are not translated into changes in secreted MMP activity. Estrogen influences on gene expression of cytokine and MMPs in primary cultures are different from those in SV40 HCEs.