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. Author manuscript; available in PMC: 2010 Jan 28.
Published in final edited form as: Genes Cells. 2009 Sep 16;14(10):1133. doi: 10.1111/j.1365-2443.2009.01340.x

Effects of Sp1 overexpression on cultured human corneal stromal cells

Xiang Shen 1, Jeong-Seok Park 1, Ye Qiu 1, Joel Sugar 1, Beatrice Y J T Yue 1,*
PMCID: PMC2813039  NIHMSID: NIHMS170883  PMID: 19758310

Abstract

Sp1, a transcription factor, is upregulated in keratoconus, a cornea-thinning disease. Keratoconus corneas have also been shown to contain increased levels of degradative enzymes such as cathepsin B and decreased proteinase inhibitors such as α1-proteinase inhibitor (α1-PI). We transfected cultured human corneal stromal cells to overexpress Sp1. The resulting effects on cathepsin B and α1-PI levels as well as the cellular proliferative and apoptotic activities were examined by Western blotting and cytochemical staining. It was found that the Sp1 transfected cells contained a greater amount of cathepsin B than did mock transfected controls. The activity of cathepsin B was also increased. By contrast, the protein level of α1-PI was lowered in corneal stromal cells upon Sp1 overexpression. The Sp1-induced alterations thus mimicked closely those observed in keratoconus, supporting the notion that Sp1 upregulation may be a key factor contributing directly to the disease development. Furthermore, the apoptotic activity was unaffected in Sp1 transfectants but the proliferation was inhibited, consistent with the idea that Sp1 may play a role in differentiation of corneal cells.

Introduction

Sp1 transcription factor belongs to a large specificity protein/Kruppel-like factor (Sp/KLF) family, sharing sequence homology and structural similarities with 25 other members (Cook et al. 1999; Suske et al. 2005; Wierstra 2008). All Sp/KLF factors contain three highly conserved C2H2-type zinc fingers, interacting with GC/GT boxes in the promoter or enhancer region of many housekeeping and inducible genes (Cook et al. 1999; Suske et al. 2005; Wierstra 2008). Ubiquitously expressed, Sp1 is involved in virtually all facets of cellular functions (Cook et al. 1999; Li et al. 2004; Suske et al. 2005; Wierstra 2008). Of particularly importance is the Sp1 regulation of TATA-less promoters that control cell growth, development, differentiation and apoptosis (McClure et al. 1999; Kavurma & Khachigian 2003; Wierstra 2008). Studies of Sp1 knockout mice further revealed that Sp1 is essential for normal mouse embryogenesis (Marin et al. 1997).

Sp1 overexpression has been associated with tumorigenesis (Safe & Abdelrahim 2005) and neuro-degenerative diseases such as Huntington (Qiu et al. 2006). The Sp1 level has also been found elevated in the cornea in a noninflammatory ocular condition called keratoconus (Whitelock et al. 1997b). Characterized by thinning, scarring and the eventual protrusion of the central portion of the cornea (Krachmer et al. 1984; Rabinowitz 1998), this corneal disease affects approximately 1 in 2–10 000 of people in the general population and leads to visual handicap in the productive second and third decades of life (Krachmer et al. 1984; Rabinowitz 1998). Its exact cause is still unclear although the pathogenesis may involve genetic along with environmental and behavioral factors (Rabinowitz 1998, 2003). No specific treatment exists, except corneal surgery when the patient's vision is beyond correction with contact lenses.

In keratoconus, the stroma, the major portion of the cornea, is the site where thinning and scarring occurs. The corneal stroma and the epithelium obtained from keratoconus patients have been shown to exhibit biochemical abnormalities in expression levels of both degradative enzymes and protease inhibitors (Sawaguchi et al. 1990, 1994; Kenney et al. 1994; Zhou et al. 1998; Brookes et al. 2003). Specifically, the levels of enzymes including cathepsins B and G (Zhou et al. 1998; Brookes et al. 2003) are markedly increased while those of inhibitors such as α1-proteinase inhibitor (α1-PI) and α2-macroglobulin are decreased (Sawaguchi et al. 1990, 1994). As a result, the degradative process in the cornea may be aberrant and the aberration has been suggested to be a mechanism underlying the development of keratoconus (Zhou et al. 1998).

The up- or down-regulation of the enzyme and inhibitor genes was noted at both protein and mRNA levels (Whitelock et al. 1997a). In view of the multiple gene involvement and the possibility of a coordinated gene regulation mechanism, several transcription factors were examined. Among them, Sp1 was found specifically upregulated in keratoconus corneas (Whitelock et al. 1997b). Studies from our laboratory have in addition demonstrated that upregulation of Sp1 suppresses the promoter activity of the human α1-PI gene in corneal cells (Li et al. 1998; Maruyama et al. 2001).

In this study, cultured cells derived from human corneal stroma were transfected to overexpress Sp1 to evaluate the resultant effects on levels of cathepsin B and α1-PI, and the cellular proliferative and apoptotic activities.

Results

The Sp1 expression vector pEGFP-Sp1 was introduced into cultured human corneal stromal cells. The level of Sp1 transcript was markedly higher (Fig. 1a). The protein level of Sp1 in the nuclear extract was also markedly enhanced (Fig. 1b). The GFP-Sp1-fusion protein expressed was localized mainly in the nuclei of the transfected cells (Fig. 2b).

Figure 1.

Figure 1

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RT-PCR (a) and Western blot (b) analyses of Sp1 transcript and protein levels in mock and pEGFP-Sp1-transfected corneal stromal cells.

Figure 2.

Figure 2

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(a) Western blotting of cathepsin B protein in mock and pEGFP-Sp1-transfected corneal stromal cells. The cathepsin B protein level was normalized against that of glyceraldehyde 3-phosphate dehydrogenase. (b) Staining for cathepsin B activity (red) in the cytoplasm of transfected (green) as well as nontransfected cells. The nuclei of all cells were stained with DAPI in blue. Note the nuclear expression of GFP-Sp1 in the transfectants. The same micrographs on the top panel are shown on the bottom without the green color to highlight the red staining for cathepsin B activity. Bar, 20 μm.

Western blotting detected a major cathepsin B protein band in corneal stromal cell lysates (Fig. 2a). This 31-kDa band appeared to correspond to the mature single-chain form processed from the inactive precursor pro-cathepsin B (Sentandreu et al. 2003). The cathepsin B level in Sp1-overexpressing stromal cells was three to fourfold higher than that in mock controls. However, considering the transfection efficiency (between 15% and 20%), the differences in the levels of cathepsin B between Sp1-transfected and non- or mock-transfected cells were even more significant. Staining for cathepsin B activity was also stronger in GFP-Sp1-expressing transfectants (Fig. 2b).

A 53-kDa protein band immunoreactive to anti-α1-PI was found in the culture medium. Its level was lowered by approximately 50% upon Sp1 transfection (Fig. 3).

Figure 3.

Figure 3

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Western blot analysis of α1-PI protein in media of cultured mock and pEGFP-Sp1- transfected corneal stromal cells. The α1-PI protein level in cell lysates was minimal (not shown).

Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase, has been used as a mitotic marker (Kee et al. 2002). Staining for Ki-67 (Fig. 4a) and the subsequent cell counting indicated that the percentage of Ki-67-positive proliferating cells was significantly (P < 0.01) reduced after Sp1 transfection (Fig. 4b).

Figure 4.

Figure 4

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Proliferative activity in mock and pEGFP-Sp1-transfected corneal stromal cells. (a) Nuclear staining for Ki-67 (pink) in GFP mock and pEGFP-Sp1-transfected (green) and nontransfected cells. All nuclei were stained with DAPI in blue. The same micrographs on the top panel are shown on the bottom only in pink and blue. Bar, 20 μm. Arrows denote Ki-67 staining in transfected cells. (b) Bar graph presenting percentages of Ki-67-positive, proliferating cells. The values were calculated based on total number of pEGFP-Sp1- or GFP mock-transfected green cells, not the total number of all DAPI-stained cells. The percent of proliferating cells in Sp1-transfected cells (32.6 ± 6.2) was significantly (P < 0.01, n = 20, asterisk) lower compared to GFP controls (51.8 ± 5.9). The percent value calculated for DAPI-stained nontransfected (non-green) cells was 59.0 ± 10.3.

While staurosporine induced a dramatic increase in percent of apoptotic cells, the Sp1 transfection showed little effect. The apoptotic level was low in GFP control, pEGFP-Sp1-transfectants, as well as nontransfected cells (Fig. 5). The percentage of ssDNA-positive apoptotic cells was 1.05 ± 0.13 (n = 20) in GFP control, 1.11 ± 0.16 in Sp1-transfected, and 61.7 ± 8.5 in staurosporine-treated corneal stromal cells.

Figure 5.

Figure 5

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Apoptotic activity in mock and pEGFP-Sp1-transfected corneal stromal cells. (a) Nuclear staining for ssDNA (pink) in transfected (green) and nontransfected cells. All nuclei were stained with DAPI in blue. Cells treated with staurosporine serve as positive controls. Bar, 20 μm. (b) Bar graph showing the percentage of ssDNA-positive apoptotic cells, which amounts to 1.05 ± 0.13 (n = 20) in GFP control, 1.11 ± 0.16 in Sp1-transfected, and 61.7 ± 8.5 in staurosporine-treated stromal cells.

Discussion

The present study demonstrates that Sp1 overexpression in human corneal stromal cells results in an elevated level and activity of cathepsin B but a decreased protein level of α1-PI.

α1-PI, a member of the serpin superfamily of proteinase inhibitors, is abundantly present in the human serum (Goodwin et al. 1997; Parfrey et al. 2003). The liver is the predominant site of its synthesis. α-PI is also found synthesized in the cornea (Twining et al. 1994). In an earlier study, we cloned and sequenced a 2.7-kb region of human α1-PI gene upstream of the corneal transcription start site. Transient transfection experiments showed that the 2.7-kb 5′-flanking DNA is functional in human corneal stromal cells and that the proximal 1.4-kb fragment is sufficient for full promoter activity (Li et al. 1998). Co-transfection of Sp1 expression vector with the 1.4-kb fragment in both corneal stromal and epithelial cells suppressed the α1-PI promoter activity (Li et al. 1998; Maruyama et al. 2001). The data obtained in the current study again support the notion that Sp1 is a negative regulator of the human α1-PI gene in corneal cells.

Cathepsin B, a lysosomal caroboxydipeptidase that has been shown to be released to the extracellular milieu and is capable of digesting gelatin and extracellular matrix elements (Buck et al. 1992), may be responsible for the abnormal degradative processes in keratoconus. The human cathepsin B promoter also possesses high GC content and lacks canonical TATA and CAAT boxes (Yan et al. 2000). Such a TATA-less promoter is often activated by Sp1 and is dependent on the presence of clusters of Sp1 binding sites in the proximity of the transcription start site (Cook et al. 1999; Suske et al. 2005; Wierstra 2008). A total of nine Sp1 binding sites are identified in the 0.44-kb fragment used previously (Maruyama et al. 2001) and cathepsin B appeared to be a typical Sp1-regulated gene. Yet the activity of the 0.44-kb cathepsin B promoter was found not affected by Sp1 co-expression in corneal cells (Maruyama et al. 2001). It was concluded then that cathepsin B expression might not be regulated directly by Sp1. The present data, however; indicated otherwise that indeed cathepsin B is upregulated by Sp1 overexpression. It is possible that the Sp1 binding site is located beyond the 0.44-kb fragment used earlier or that additional co-factors are required for the Sp1 regulation.

Overexpression of Sp1 retards proliferation in corneal stromal cells. This retardation may be a factor contributing to the lowered keratocyte density in keratoconus patients who wear contact lenses (Erie et al. 2002). Previously in smooth muscle cells, an antimitogenic, pro-apoptotic role of Sp1 has been demonstrated by repression at the level of p21WAF1/Cip1 transcription, mRNA and protein (Kavurma & Khachigian 2003). The Sp1 phenotype manifested nevertheless is highly context- and cell type dependent (Wierstra 2008). For instance, overexpression of Sp1 in vascular endothelial cells, in direct contrast to smooth muscle cells, activates p21WAF1/Cip1 and stimulates cell proliferation (Gartel & Tyner 1999). In corneal stromal cultures, Sp1 overexpression does not affect the apoptotic activity but inhibits cell proliferation. This suggests that Sp1 may have a role in differentiation of corneal cells.

It is of note that the corneal stromal cells cultured with serum in our laboratory were corneal fibroblasts, morphologically and functionally different from the native keratocytes. The expression of keratocyte gene marker keratocan is altered in corneal fibroblasts (Funderburgh et al. 2003). These corneal cells, however, still retain some of the inherent characteristics, producing for instance a small amount of keratan sulfate and are distinct from cutaneous fibroblasts (Klint-worth & Smith 1981). Furthermore, keratoconus fibroblasts have been shown previously to contain a hightened basal level of Sp1 compared to those from normal corneas, implying that the diseased corneal cells do carry and retain the Sp1 abnormality even after being maintained in serum-containing tissue culture media (Cheng et al. 2001).

Taken together, Sp1 overexpression leads to an increase in both the level and activity of cathepsin B in cultured corneal stromal cells. The α1-PI level is by contrast decreased. The Sp1-induced alterations demonstrated in the current study seem to mimic closely those observed in keratoconus corneas. This suggests that the Sp1 upregulation noted in keratoconus may mediate and contribute directly to the events leading to the pathology. Using Sp1-siRNA or another approach to suppress Sp1 may result in downregulation of cathepsin B and upregulation of α1-P1, providing potentially a therapeutic modality for keratoconus.

While it is still unclear why Sp1 is upregulated in keratoconus, earlier developmental studies (Nakamura et al. 2005, 2007) revealed that the expression of Sp1 in the cornea is temporally controlled. Both the Sp1 protein and transcript are abundant in the cornea at embryonic stages, but their levels are substantially reduced after eyelid opening in the mouse (Nakamura et al. 2005) or shortly after birth in human (Nakamura et al. 2007). Perhaps in keratoconus, the postdevelopmental program engineered to silence Sp1 is aberrant such that the Sp1 level in the mature cornea remains abnormally high or unsuppressed. Epigenetics and/or the ubiquitin-proteasome system may be the possible candidates involved in the homeostatic Sp1 silencing.

Experimental procedures

Cell culture

Normal human eyes from donors 12, 17, 23 and 32 years of age were obtained from the Illinois Eye Bank (Chicago, IL, USA). The procurement of tissues was approved by the Institutional Review Board at the University of Illinois at Chicago in accordance with the Declaration of Helsinki. The corneal stromal tissues were dissected and cultured as previously described (Yue & Baum 1981) in complete media that contained Dulbecco's modified Eagle's minimum essential medium and fetal bovine serum. The corneal stromal cells have a fibroblastic morphology and are also often referred to as corneal fibroblasts. Second- or third-passaged cells were used for the study.

Sp1 constructs and transfection into corneal stromal cells

Human Sp1 was amplified by PCR using 5′-GCCGGTACCGTGAAGCCATTGCCACTGATATTAATGG-3′ and 5′-GCCCTCGAGCCACCATGAGCGACCAAGATC-3′ primers. The 2.4-kilobase (kb) Sp1 PCR product was subcloned into pEGFP-N1 (BD Biosciences, Lexington, KY, USA) to yield pEGFP-Sp1.

pEGFP-Sp1 was introduced into human corneal stromal cells using FuGENE 6 (Roche, Indianapolis, IN, USA). As mock controls, cells were also transfected in parallel with pEG-FP-N1 empty vector without the insert.

Reverse transcription-PCR

cDNA was prepared from total RNA extracted from stromal cells using random hexamers and the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). PCR for Sp1 was performed using primers 5′-GTGGCAATAAT GGGGGCAATG-3′ and 5′-GGCAACTACCCGTTGTCGAC-3′. For 18S ribosomal RNA (rRNA), the universal primers from Ambion (Austin, TX, USA) were used. PCR products were resolved on 1% agarose gels. The expected sizes for Sp1 and 18S rRNA products are 497 and 315 base pairs, respectively. Densitometry was performed and the band intensity of Sp1 product was normalized to that of 18S rRNA.

Western blot analysis

Corneal stromal cells were lysed in buffer containing protease inhibitors (Roche). Nuclear extraction was carried out using a nuclear extraction kit (Activ Motif, Carlsbad, CA, USA). The proteins in the extracts or the total lysates were quantified by Bradford protein assay (Bio-Rad, Hercules, CA, USA). For α1-PI, the media were also collected after incubating the transfected cells with serum-free media for additional 24 h. Equal aliquots of extracts, lysates or media (20 μg protein equivalent) were subjected to 10% sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions and immunoblotted with polyclonal anti-Sp1 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-cathepsin B (Calbiochem, San Diego, CA, USA), anti-α1-PI (MP, Santa Ana, CA, USA) or anti-glyceraldehyde 3-phosphate dehydrogenase (Trevigen, Gai-thersburg, MD, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel, Irvine, CA, USA) was used as the secondary antibody. The intensity of protein bands visualized was measured by densitometry.

Cathepsin B activity

The activity of cathepsin B in transfected cells was detected using the Magic Red cathepsin B kit (Immunochemistry Technologies, Bloomington, MN, USA).

Ki-67 staining

Transfected cells were fixed in cold methanol and permeabilized. After blocking, cells were incubated for 1 h with anti-Ki-67 (Zymed, South San Francisco, CA, USA) which stains cells that have entered into the cell cycle, or normal mouse IgG (negative control). Following incubation with FITC-goat anti-mouse IgG (Jackson Immuno Research, West Grove, PA, USA), slides were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) which stains all cell nuclei independent of cell cycle stage. Both were examined. The total number of GFP- or GFP-Sp1-overexpressing green cells and the number of Ki-67-positive transfectants in 20 of 10× fields selected in a randomized manner were counted. The percent of Ki67-positive proliferating cells in the transfected cells was calculated.

Apoptosis

Cells after transfection were fixed, permeabilized, and allowed to react with anti-single-stranded (ss)DNA (Alexis, San Diego, CA, USA) and Cy3-conjugated goat anti-mouse IgM (Jackson Immuno Research). As a positive control, nontransfected corneal stromal cells were treated with 1 μM staurosporine for 4 h prior to the procedure. The total number of DAPI-stained transfected cells and the number of ssDNA-positive green cells in 20 fields selected in a randomized manner were determined. All experiments were repeated at least three times. Statistical analysis was performed using the Student's t-test.

Acknowledgments

We thank Ms Ruth Zelkha for assistance in image analysis. This work was supported by grants EY03890 and EY05628 (B.Y.J.T.Y.) and core EY01792 from the National Eye Institute, a grant from Center for Keratoconus, and a Senior Scientific Investigator Award (B.Y.J.T.Y.) from Research to Prevent Blindness, Inc.

Footnotes

Communicated by: Noriko Osumi

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