|Molecular Vision 2007;
Received 18 April 2007 | Accepted 17 August 2007 | Published 27 August 2007
Expression of Sp1 and KLF6 in the developing human cornea
Deepak P. Edward,
Joel Sugar, Beatrice
Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, IL
Correspondence to: Hiroshi Nakamura, M.D., Ph.D., Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, Chicago, IL, 60612; Phone: (312) 355-3799; FAX: (312) 996-7773; email: firstname.lastname@example.org
Purpose: To examine the temporal and spatial expression of Sp1 and Krüppel-like factor 6 (KLF6) in the cornea in fetal and adult human eyes.
Methods: Eyes from human fetus (F) of 7, 9, 10, 11, 13, and 27 weeks (w) of gestation, as well as corneas from 11 and 56-day (d)-old children and donors 2, 6, 16, 25, 40, 51, 69, and 83 years (y) of age were obtained. All specimens were fixed in 10% buffered formalin, processed for paraffin sections, and examined for Sp1 and KLF6 expression immunohistochemically.
Results: Staining for Sp1 was evident at the earliest F7w time point in the cornea. From F7w to F27w, the moderate to strong Sp1 immunostaining was seen in the nuclei of epithelial and endothelial cells. Staining in keratocytes was also observed. The intensity of Sp1 staining in all layers of the cornea was substantially decreased 11d after birth and remained low thereafter. Positive KLF6 staining was also noted at F7w in all corneal layers. In the epithelium and endothelium, the staining was mostly cytoplasmic throughout the fetal stages. After birth, the KLF6 staining appeared in the nuclei of corneal epithelial cells along with that in the cytoplasm. The intensity of KLF6 staining in the epithelium and endothelium remained relatively constant from E47d to the 83y-old donor cornea. The KLF6 staining in the stroma however was reduced after F27w.
Conclusions: The present study indicates that the expression of Sp1 and KLF6 is developmentally regulated, providing a basis for further investigations on the regulation of the Sp1 and KLF6 gene during the course of corneal development and in corneal diseases such as keratoconus.
Sp1 and Krüppel-like factor-6 (KLF6, also known as Zf9/CPBP) are members of a growing family of SP/KLF transcription factors that currently has 26 known members (Sp1-9 and KLF1-17) . Transcription factors are known to bind specific promoter regions of various genes to either enhance or repress transcription of a particular gene by assisting or blocking RNA polymerase binding . Sp1 and KLF6 proteins are characterized by homologous zinc-finger domains close to their COOH-terminus. The NH2-terminal regions, by contrast, are highly variable. All Sp/KLF factors interact via zinc fingers with GC(CACCCC) boxes in the promoter elements of many cellular genes. The physiological roles of the Sp/KLFs are diverse.
Sp1, one of the first eukaryotic transcription factors identified, was originally cloned as a factor that binds to the SV40 early promoter [3,4]. Ubiquitously expressed , this nuclear protein has been implicated in the activation or suppression of a large number of genes, and is shown to be involved in cellular processes such as cell cycle regulation [6-8], chromatin remodeling [9,10], prevention of CpG island methylation [11,12], and apoptosis [8,13-15]. Sp1 knockout mice further revealed that Sp1 is essential for normal mouse embryogenesis. All Sp1-/- mice die around day 11 of gestation .
KLF6 was originally cloned from cDNA libraries of placenta. Human KLF6 mRNA is ubiquitously expressed, with a high level of expression in the liver, lung, intestine, prostate, and placenta [17,18]. KLF6 has been shown to play a crucial role in regulation of genes involved in tissue development, differentiation, angiogenesis, hematopoiesis, cell cycle control, and proliferation . KLF6 and other KLF family member such as KLF4, are also shown to be expressed and/or important for eye development [20,21].
Sp1 and KLF6 are both expressed in the human cornea [22,23]. In vitro experiments with corneal epithelial cells have shown that Sp1 upregulates involucrin  and α5 integrin subunit through fibronectin , and KLF6 upregulates keratin 12 promoter activity . Our previous studies have shown that Sp1 is specifically upregulated in keratoconus, a noninflammatory disease characterized by thinning, scarring, and the eventual protrusion of the central portion of the cornea . An increase in KLF6 expression has also been found in some keratoconus cases . Gene regulation of these transcription factors in relation to the development of keratoconus however remains elusive.
We have previously examined the expression of Sp1 and KLF6 in mouse eyes [20,28], and shown that these factors, especially Sp1, may be important for corneal development in mouse. Sp1 expression in the cornea appears to be temporally regulated. It is abundant in the embryonic cornea and also in postnatal stages, but is much reduced after eyelid opening . KLF6, on the other hand, is expressed throughout mouse corneal development . Its subcellular localization is switched from predominantly cytoplasmic in the embryonic cornea to both nuclear and cytoplasmic in postnatal stages.
The current study was undertaken to extend our efforts to humans, investigating the temporal and spatial expression of Sp1 and KLF6 in the cornea from fetal stages to adult.
The study was approved by the Institutional Review Board at the University of Illinois at Chicago. Eyes from human fetus (F) of 7, 9, 10, 11, 13, and 27 weeks (w) of gestation were obtained from the University of Washington Birth Defects laboratory (Seattle, WA). Corneas from an 11-day (d)-old child and donors 2, 6, 16, 25, 40, 51, 69, and 83 years (y) of age, were obtained from Illinois Eye Bank (Chicago, IL) or the National Disease Research Interchange (Philadelphia, PA). None of the eyes had any records of known ocular diseases, and all corneas were clear and unremarkable.
All fetus eyes and corneas were fixed in 10% buffered formalin within 4 and 24 h following death, respectively, and processed subsequently for paraffin sections. For immunohistochemistry, 5 μm-thick sections were deparaffinized and rehydrated. After blocking with goat serum (1:10), sections were incubated at room temperature sequentially with polyclonal rabbit anti-Sp1 (1:100, PEP2; Santa Cruz Biotechnology, Santa Cruz, CA) or polyclonal rabbit anti-KLF6 (1:100, R-173; Santa Cruz Biotechnology) for 90 min. Biotinylated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA) was used as a secondary antibody for a further 45 min, room temperature incubation. For KLF6 immunostaining, the sections were boiled with sodium citrate buffer (10 mM, pH 6.0, NeoMarkers, Fremont, CA) for 10 min for antigen retrieval prior to the blocking procedure . For Sp1 detection, the sections were incubated with alkaline phosphatase conjugated extravidin (1:50; Sigma, St. Louis, MO) and the color reaction was carried out using Fast Red TR/Naphthol AS-MX Phosphate (Sigma) for 5 min. For KLF6, sections were incubated at room temperature with dichlorotriazinyl aminofluorescein (DTAF)-conjugated extravidin (Jackson ImmunoResearch) for 45 min. Specimens were then mounted in aqueous mounting fluid containing 4',6 diamidino-2-phenylindole (DAPI) which produces a blue fluorescence as counterstaining of the nucleus (Vectashield, Vector Laboratories, Burlingame, CA). Staining intensity for Sp1 and KLF6 protein was graded on a per cell basis under a light and fluorescence microscope, respectively, by three observers. Minus signs indicate no staining, and "±" to "+++" indicates increasingly intense staining . Experiments were repeated twice.
For comparison purposes, images of all specimens were captured with a Zeiss Axioscope 2 and Axiovision software 4.2 (Carl Zeiss MicroImaging, Thornwood, NY) under the same exposure time. In addition, deconvolution images were acquired using a Zeiss Axiovert 100 microscope (Carl Zeiss MicroImaging) with the aid of MetaMorph (Molecular Devices, Downingtown, PA) and AutoQuant (version 9 and 10; Meyer Instruments, Houston, TX) software.
Immunostaining for Sp1 in the corneal epithelium, stroma, and endothelium was evident at the earliest F7w stage (Figure 1; F7w). From F7w to F27w, the moderate to strong Sp1 immunostaining was seen mainly in the nuclei of epithelial and endothelial cells (Figure 1, F7w-F27w and deconvolution images). Moderate staining in keratocytes was also observed in this time span, although it was difficult to discern whether the staining was nuclear or cytoplasmic (Figure 1; F7w-F27w). The intensity of Sp1 staining in all layers of the cornea was substantially decreased at the 11 days after birth time point, and remained low thereafter (Figure 1; 11d-69y).
Positive staining for KLF6 was also noted at F7w in all corneal layers (Figure 2; F7w). In the epithelium and endothelium, the KLF6 staining was mostly cytoplasmic at all the fetal stages studied. After birth, staining appeared in the nuclei of corneal epithelial cells along with that in the cytoplasm (Figure 2; 11d-69y, and deconvolution images). The intensity of KLF6 staining in the epithelium and endothelium remained relatively constant from E47d to the 83y-old donor cornea. The KLF6 staining in the stroma, however, was reduced after F27w (Figure 2; F27w-69y).
Staining intensity for Sp1 and KLF6 in the basal corneal epithelium, stroma, and endothelium at each developmental stage point was scored. The range of scores is summarized in Figure 3. Sp1 had a declining nuclear expression pattern, and KLF6 staining showed a translocation from cytoplasm to nucleus.
The present study examines the expression pattern of transcription factors Sp1 and KLF6 in the developing human cornea. Because of the scarcity of fetal corneal tissues, we studied specimens from only 6 fetal stages and one from an 11d child. Nevertheless, our results demonstrate that the expression of Sp1 is temporally regulated in the developing human cornea. The Sp1 protein in corneal basal epithelial cells, keratocytes, and endothelial cells was abundant during fetal stages. It was then dramatically decreased after birth. These observations are consistent with our previous findings on mouse corneas  that Sp1 expression was substantially downregulated after eyelid opening. Along with other developmental  and Sp1 null mouse  studies in which Sp1 was found upregulated in developing cells and downregulated in fully differentiated cells, our data support the notion that Sp1 has a critical regulatory role during early corneal development.
KLF6, by contrast, appears to be spatially regulated. The level of this transcription factor was rather constant throughout the stages examined. The subcellular localization of KLF6 was mostly in the cytoplasm in fetal stages in the human corneal epithelium and endothelium. Nuclear KLF6 appeared after birth, and the KLF6 localization remained nuclear throughout the subsequent stages. Such a translocation, which may be related to events at birth such as altered light and exposure to oxygen, was also seen in our previous mouse study , that nonnuclear staining of KLF6 was observed in the mouse corneal epithelium from E10.5d to postnatal day 7 while the nuclear staining was abundant in stages between E18.5 and postnatal day 60. These data are also in keeping with findings from other cell types including hepatic and arterial endothelial cells that KLF6 has a preferential distribution in the cytoplasmic compartment and that nuclear translocation of KLF6 occurs upon treatment of stimulus or after injury during repair [30-32]. The observation that KLF6 nuclear localization is detected in the cornea after birth seems to suggest that KLF6 plays a minor role during corneal development. However, the biological context of this observation is still likely to be important. A recent study using conditional knockout mouse has demonstrated that another member of the KLF family, KLF4, is critical in postnatal maturation and maintenance of the ocular surface, including the cornea . KLF6 may have a postnatal maturation role similar to that of KLF4.
Sp1 has been shown to be upregulated in keratoconus. While the mechanism is unclear, results of the present study imply that, perhaps in keratoconus, the post-developmental 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 possible candidates involved in the homeostatic Sp1 silencing. There are also other noninflammatory diseases characterized by corneal thinning, such as keratoglobus, pellucid marginal degeneration, and ketatectasia. Investigation of Sp1 expression in those diseases may be of interest and may help define their relationship with keratoconus. We have, at this point, found that the Sp1 expression in keratectasia after LASIK is, unlike in keratoconus, not abnormally upregulated .
There may, in addition, be more widespread significance to the current Sp1 findings. For example, the expression and/or localization of protein related to development such as Sp1 may have relevance and help dissect the healing processes in matured tissues. Interestingly, it has been reported that Sp1 is expressed during the wound healing process in vivo in the corneal epithelium . Sp1 expression and its binding activity to target sites of DNA have also been demonstrated to be increased considerably in in vitro corneal epithelial cells grown on flasks coated with fibronectin, which is transiently expressed at high levels during wound healing of the corneal epithelium [25,35].
Our previous studies have suggested that KLF6 is increased in some keratoconus corneas, contributing perhaps also to the pathophysiology. The increased KLF6 may act independently or cooperate with Sp1 to cause or escalate pathology. Direct physical interaction between KLF6 and Sp1 has been documented by biochemical experiments and one hybrid system , and these two factors have been shown to cooperatively regulate the expression of genes including endoglin and collagen α1 (I) .
In summary, we presented herein a dynamic pattern of developmental expression of transcription factors Sp1 and KLF6 in the human cornea. The results provide a basis for further investigations on regulation of the Sp1 and KLF6 gene both during corneal development and in diseases such as keratoconus.
This research was supported by grants EY03890 (B.Y.J.T.Y.), EY05628 (B.Y.J.T.Y.), and a core grant EY01792 from the National Institutes of Health, Bethesda, MD. Further support came from an unrestricted grant from Research to Prevent Blindness, New York, NY and a gift from Ralph Lindauer.
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