Elsevier

Experimental Eye Research

Volume 85, Issue 4, October 2007, Pages 539-545
Experimental Eye Research

Hyaluronan production regulation from porcine hyalocyte cell line by cytokines

https://doi.org/10.1016/j.exer.2007.07.006Get rights and content

Abstract

The objective of this study were to establish a cell line derived from porcine hyalocytes and to investigate the regulation of hyaluronan (HA) synthesis in response to cytokines. After 50 passages of the cells derived from porcine vitreous tissue, a cell line was generated. The immortalized cells showed fibroblastic morphology. The cell doubling time was 56.9 h. In the mRNA level, the cells expressed plate-derived growth factor (PDGF) α receptor, PDGF β receptor, transforming growth factor-β (TGF-β) type I receptor, TGF-β type II receptor, CD44, collagen type I, collagen type II, glial fibrillary acidic protein (GFAP), hyaluronan synthase (HAS) 2, HAS 3 and β-actin. In the protein level, GFAP was expressed in this cell line. S-100 protein and cytokeratin were not detected. Stimulation with TGF-β1 and/or PDGF-BB induced a marked increase in the expression level of HAS2 mRNA, and induced HA production. TGF-β1 stimulated HAS2 expression through the signal transduction pathway including Smad 2,3,4. In summary, this report constitutes the first successful immortalization of porcine hyalocyte cells. The production of HA was induced from the generated porcine hyalocyte cell line under the stimulation of TGF-β1 and/or PDGF-BB, which may be related to the pathogenesis of proliferative membrane formation in proliferative vitreo-retinal diseases.

Introduction

Hyaluronan (HA), an extracellular matrix (ECM) component, is a high molecular weight linear glycosaminoglycan composed of repeating disaccharide units with the following structure: d-glucuronic acid β(1  3) N-acetylglucosamine β(1  4) (Laurent and Fraser, 1992). HA is an important constituent of ECM and plays an essential role in regulating cell migration, proliferation, adhesion, development, and differentiation (Laurent and Fraser, 1992; Knudson and Knudson, 1993). HA synthesis is catalyzed by membrane-bound enzymes, termed hyaluronan synthases (HAS1, HAS2, HAS3) (Weigel et al., 1997). Each HAS isoform possesses the ability to synthesize HA molecules of a given size, exhibit different kinetic properties, and a cell type specific pattern (Weigel et al., 1997, Itano et al., 1999, Brinck and Heldin, 1999). The expression levels of each HAS gene and protein are regulated differently by several cytokines and growth factors, such as platelet derived growth factor-BB (PDGF-BB) and transforming growth factor-β (TGF-β) (Heldin et al., 1989, Heldin et al., 1992, Suzuki et al., 1995, Sugiyama et al., 1998, Usui et al., 1999, Usui et al., 2000, Usui et al., 2003, Jacobson et al., 2000).

We previously reported the HAS expression by the resident cells of proliferative membranes in vitreo-retinal diseases, suggesting a potential role in the pathogenesis of the disease (Suzuki et al., 2003). Recent studies (Sakamoto, 2003, Noda et al., 2004) indicate that hyalocytes are concerned in intraocular proliferative diseases with investigation of bovine hyalocytes under PDGF-rich condition. Collectively, the evidence accumulated suggest that studies on the molecular mechanisms that regulate HA synthesis by hyalocytes may be useful in understanding the physiological significance of the interaction between ECM and growth factors in intraocular proliferative diseases.

The previous study (Andley et al., 1994) constituted the first successful immortalization of human lens epithelial cells with the SV40-T antigen gene. Cell lines can provide a constant and reproducible source for the research.

In the present study, we established the cell line derived from vitreous cells and investigated the regulation of HA synthesis in response to growth factors to gain further insight into the pathogenesis of intraocular proliferative diseases.

Section snippets

Isolation, culture, immortalization and growth of porcine vitreous-derived cell line

Two year old porcine eyes were obtained from a slaughterhouse. Vitreous body was dissected carefully with microscope as follows. The cornea was removed with forceps and vitreous body and lens were extracted with compression the eyeball. The extracted vitreous body was chopped into several pieces and placed on a 100 mm well culture plate (CELLSTAR; greiner bio one, Germany) in Dulbecco's modified Eagle's medium (DMEM; Gibco, NY, USA) with 10% fetal bovine serum (FBS) and 100 U/ml penicillin, 100 

Establishment of the porcine vitreous-derived cell line

To culture the vitreous cells selectively, we observed the extracted vitreous body. The detrited ciliary body adhered to compressed extracted whole vitreous body (Fig. 1A). So we could observe the vitreous body more anatomically. There were a lot of round cells near pars plana in finding of phase contrast microscope (Fig. 1B). We selectively extract this area carefully and cultured.

Transfection of SV-40T gene into primary culture cells resulted in one cell line. Previous studies have

Discussion

In the present study, we established the PH5 cell line derived from porcine vitreous cells. Both primary cells and PH5 cells have similar character in our investigation (Fig. 3). PH5 cells express GFAP which is recognized as a marker for astrogrial cells in the normal human retinae (Dreher et al., 1992, Madigan et al., 1994). S-100 protein and cytokeratin were not detected in both cells. Previous report (Noda et al., 2004) showed that the hyalocytes were immunocytochemically positive for S-100

References (42)

  • P.H. Weigel et al.

    Hyaluronan synthases

    J. Biol. Chem.

    (1997)
  • M. Zhu et al.

    The human hyaloid system: cellular phenotypes and inter-relationships

    Exp. Eye Res.

    (1999)
  • U.P. Andley et al.

    Propagation and immortalization of human lens epithelial cells in culture

    Invest. Ophthalmol. Vis. Sci.

    (1994)
  • P. D'Amore

    Mechanisms of retinal and choroidal neovascularization

    Invest. Ophthalmol. Vis. Sci.

    (1994)
  • R. Deed et al.

    Early-response gene signalling is induced by angiogenic oligosaccharides of hyaluronan in endothelial cells. Inhibition by non-angiogenic, high-molecular-weight hyaluronan

    Int. J. Cancer

    (1997)
  • Z. Dreher et al.

    Muller cells in vascular and avascular retinae: a survey of seven mammals

    J. Comp. Neurol.

    (1992)
  • R.N. Frank

    Vascular endothelial growth factor—its role in retinal vascular proliferation

    N. Engl. J. Med.

    (1994)
  • P. Heldin et al.

    Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts

    Biochem. J.

    (1989)
  • P. Heldin et al.

    Characterization of the molecular mechanism involved in the activation of hyaluronan synthase by platelet-derived growth factor in human mesothelial cells

    Biochem. J.

    (1992)
  • C.H. Heldin et al.

    TGF-β signaling from cell membrane to nucleus through SMAD proteins

    Nature

    (1997)
  • A. Jacobson et al.

    Expression of human hyaluronan synthases in response to external stimuli

    Biochem. J.

    (2000)
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