Characterization of the human α9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells.

α9ß1 is the most recent addition to the integrin family of membrane receptors and consequently remains the one that is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the α9 gene. Most of all, addition of tenascin-C (TNC), the ligand of α9ß1, to the tissue culture plates prior to seeding HCECs increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.

Altered expression of the poly(ADP-ribosyl)ation enzymes in uveal melanoma and regulation of PARG gene expression by the transcription factor ERM.

PURPOSE: Poly(ADP-ribosyl)ation is a reversible post-translational modification that requires the contribution of the enzymes poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Our study explores expression and activity of PARP-1 and PARG in uveal melanoma cell lines with varying tumorigenic properties. METHODS: Gene profiling on microarrays was conducted using RNA prepared from the uveal melanoma cell lines T97, T98, T108, and T115. The activity of PARP-1 and PARG was monitored by enzymatic assays, whereas their expression was measured by Western blot and PCR. The PARG promoter was analyzed using promoter deletions and site-specific mutagenesis in transfection analyses. The transcription factors binding the PARG promoter were studied by electrophoretic mobility shift assay (EMSA) analyses. Suppression of PARP-1 and PARG expression was performed in T97 and T115 cells by RNAi, and their tumorigenic properties monitored by injections into athymic mice. RESULTS: Expression of PARP-1 was found to vary considerably between uveal melanoma cell lines with distinctive tumorigenic properties in vivo. Sp1 and the ETS protein ERM were shown to bind to the PARG gene promoter to ensure basal transcription in uveal melanoma. Importantly, suppression of PARG gene expression in T97 and T115 cells increased their capacity to form tumors in athymic mice, whereas suppression of PARP-1 significantly reduced or almost entirely abolished tumor formation. CONCLUSIONS: Our results suggest that while overexpression of PARP-1 may confer a proliferative advantage to aggressive uveal melanoma tumors, PARG may, on the other hand, support a tumor suppressor function in vivo.

Suppression of α5 gene expression is closely related to the tumorigenic properties of uveal melanoma cell lines.

Cancer aggressiveness is related to the ability of cancer cells to escape the anchorage dependency toward the extracellular matrix, a process regulated by the integrin α5ß1 and its ligand fibronectin. Here, we characterized the expression of the α5 gene in human uveal melanoma cell lines with distinct tumorigenic properties and investigated some of the mechanisms underlying the variations of their malignancy. Strong and weak expression of α5 was observed in cells with no (T108/T115) and high (T97/T98) tumorigenic properties, respectively. Expression and DNA binding of the transcription factors Sp1, activator protein 1 (AP-1) (both acting as activators), and nuclear factor I (NFI) (a strong repressor) to the α5 promoter were demonstrated in all cell lines. A reduced expression of AP-1 combined with a dramatic increase in NFI correlated with the suppression of α5 expression in T97 and T98 cells. Restoring α5 expression in T97 cells entirely abolished their tumorigenicity in immunodeficient mice. These uveal melanoma cell lines might therefore prove particularly useful as cellular models to investigate α5ß1 function in the pathogenesis of invasive uveal melanoma.

Electrophoretic mobility shift assays for the analysis of DNA-protein interactions.

Electromobility shift assay is a simple, efficient, and rapid method for the study of specific DNA-protein interactions. It relies on the reduction in the electrophoretic mobility conferred to a DNA fragment by an interacting protein. The technique is suitable to qualitative, quantitative, and kinetic analyses. It can also be used to analyze conformational changes.

Differential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human alpha5 integrin gene dictates its transcriptional activity.

PURPOSE: Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor alpha5beta1. The authors reported previously that FN induces alpha5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the alpha5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on alpha5 gene expression and evaluated the contribution of FN to their overall regulatory influence. METHODS: TF binding to the alpha5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. RESULTS: Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the alpha5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the alpha5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress alpha5 promoter activity in a cell density-dependent manner by differentially altering the ratio of these TFs. CONCLUSIONS: These results suggest that alpha5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) alpha5 transcription in corneal epithelial cells.

Transcriptional regulation of the human alpha6 integrin gene by the transcription factor NFI during corneal wound healing.

PURPOSE: Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human alpha6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human alpha6 gene, and its regulatory influence was characterized in corneal epithelial cells. METHODS: Plasmids bearing the alpha6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the alpha6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. RESULTS: All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of alpha6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of alpha6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. CONCLUSIONS: Repression of alpha6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane.

Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1.

BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity. RESULTS: Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but neither with Sp3 nor NFI, in PARP-1+/+ cells. In addition, in vitro PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1+/+ and PARP-1-/- cells revealed that the lack of PARP-1 expression in PARP-1-/- cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1-/- cells. Subjecting PARP-1+/+ cells to an oxidative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function. CONCLUSION: Our results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription.

Laminin reduces expression of the human alpha6 integrin subunit gene by altering the level of the transcription factors Sp1 and Sp3.

PURPOSE: Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after corneal injury, which is later replaced by the secretion of laminin (LM). Laminin is recognized by receptors (alpha6beta1 and alpha6beta4) that belong to the integrin family. The authors characterized the regulatory influence exerted by laminin on the Sp1/Sp3-mediated alpha6 gene promoter function. METHODS: Recombinant plasmids bearing the CAT reporter gene, fused to various segments from the alpha6 promoter, were transfected into rabbit corneal epithelial cells (RCECs) grown on untreated or on LM-coated culture plates or into Sp1-deficient Drosophila Schneider cells. Expression and DNA binding of Sp1/Sp3 was monitored with the use of Western blot and electrophoretic mobility shift assays (EMSAs), respectively. DNA target sites for Sp1/Sp3 in the alpha6 gene promoter were identified in vitro by DNAse I footprinting. Binding of Sp1 and of a few other transcription factors was also examined in vivo by chromatin immunoprecipitation (ChIP) assays. Expression of the alpha6 mRNA in RCECs grown on LM-coated culture plates was also assessed by Northern blot and RT-PCR analyses. RESULTS: Transfection experiments provided evidence that both Sp1 and Sp3 positively influence alpha6 promoter activity in Sp1/Sp3-deficient SL2 Schneider cells. Two GC-rich target sites for Sp1/Sp3 (a proximal and a distal site) were identified in the alpha6 promoter by DNAse I footprinting. Binding of Sp1/Sp3 was further validated in vivo by ChIP. Transfections conducted into RCECs grown on LM-coated culture plates resulted in repression of the activity directed by the alpha6 promoter. This LM-mediated negative regulatory influence also translated into a similar reduction in the expression of the endogenous alpha6 mRNA as revealed by both RT-PCR and Northern blot analyses. Most of all, results from EMSA and Western blot analyses suggested that the LM-mediated repression of the alpha6 promoter activity results in part from the proteolytic cleavage of Sp1/Sp3. CONCLUSIONS: Unlike FN, which functions as an activator of alpha5 gene transcription, LM repressed transcription, directed by the alpha6 gene promoter, by altering the nuclear levels of Sp1 and Sp3. Reappearance of LM in the basement membrane after repair of the corneal damage is therefore expected to substantially contribute to the final steps of this process by influencing the degree to which genes that encode protein products requested for cell adhesion and migration, such as integrin genes, are expressed during corneal wound healing.

Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells.

The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via specific protein-protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considerably increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Over-expression of each of the four NFI isoforms in HCT116 cells established that each of them contribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby establishing a role for NFI in the cell cycle dependent expression of p21.

The feeder layer-mediated extended lifetime of cultured human skin keratinocytes is associated with altered levels of the transcription factors Sp1 and Sp3.

Primary cultured epithelial cells that are used for basic research are often cultivated on plastic whereas those used for clinical purposes are usually cultured in the presence of a feeder layer. Here, we examined the influence of a feeder layer on the expression, affinity and DNA binding ability of the transcription factors, Sp1 and Sp3 in primary cultures of human skin keratinocytes. Co-culturing both newborn and adult skin keratinocytes with lethally irradiated 3T3 cells as a feeder layer contributed to maintain the cell's morphological and growth characteristics and delayed terminal differentiation in vitro. 3T3 also stabilized the DNA binding properties of Sp1 without altering its transcription. Stimulation of Sp1/Sp3 expression appears to be mediated through cell-cell interactions and by factors secreted by 3T3. Thus, we propose that the feeder layer delay terminal differentiation of primary cultured skin keratinocytes by preventing extinction of transcription factors, like Sp1 and Sp3, which play pivotal functions in the cell cycle.

Regulation of the poly(ADP-ribose) polymerase-1 gene expression by the transcription factors Sp1 and Sp3 is under the influence of cell density in primary cultured cells.

PARP-1 [poly(ADP-ribose) polymerase-1) is a nuclear enzyme that is involved in several cellular functions, including DNA repair, DNA transcription, carcinogenesis and apoptosis. The activity directed by the PARP-1 gene promoter is mainly dictated through its recognition by the transcription factors Sp1 and Sp3 (where Sp is specificity protein). In the present study, we investigated whether (i) both PARP-1 expression and PARP-1 enzymatic activity are under the influence of cell density in primary cultured cells, and (ii) whether its pattern of expression is co-ordinated with that of Sp1/Sp3 at varying cell densities and upon cell passages. All types of cultured cells expressed PARP-1 in Western blot when grown to sub-confluence. However, a dramatic reduction was observed at post-confluence. Similarly, high levels of Sp1/Sp3 were observed by both Western blot and EMSAs (electrophoretic mobility-shift assays) in sub-confluent,but not post-confluent, cells. Consistent with these results, the promoter of the rPARP-1 (rat PARP-1) gene directed high levels of activity in sub-confluent, but not confluent, cells upon transfection of various CAT (chloramphenicol acetyltransferase)-rPARP-1 promoter constructs into cultured cells. The positive regulatory influence of Sp1 was not solely exerted on the rPARP-1 promoter constructs, as inhibition of endogenous Sp1 expression in HDKs(human dermal keratinocytes) through the transfection of Sp1 RNAi (RNA interference) considerably reduced endogenous hPARP-1 (human PARP-1) expression as well. The reduction in PARP-1 protein expression as cells reached confluence also translated into a corresponding reduction in PARP-1 activity. In addition, expression of both Sp1/Sp3, as well as that of PARP-1,was dramatically reduced as cells were passaged in culture and progressed towards irreversible terminal differentiation. PARP-1 gene expression therefore appears to be co-ordinated with that of Sp1 and Sp3 in primary cultured cells, suggesting that PARP-1 may play some important functions during the proliferative burst that characterizes wound healing.

Expression of the alpha4 integrin subunit gene promoter is modulated by the transcription factor Pax-6 in corneal epithelial cells.

PURPOSE: Expression of several membrane-bound integrins is thought to be altered during corneal wound healing as a consequence of the massive secretion of fibronectin occurring during this process. Examination of the alpha4 integrin subunit gene promoter revealed the presence of three putative binding sites for the transcription factor Pax-6 expressed in the basal cells of the corneal epithelium during corneal wound healing. This study was undertaken to investigate whether the alpha4 integrin subunit is expressed in primary cultures of rabbit corneal epithelial cells (RCECs) and to test whether Pax-6 binds the alpha4 gene promoter and regulates its transcriptional activity. METHODS: Both flow cytometry and immunocytochemical analyses, along with an antibody-directed receptor interference assay, were used to examine expression of the alpha4 subunit in RCECs. Expression of Pax6 was investigated by immunoblot analysis. Binding of PAX6 to the alpha4 gene promoter was tested in electrophoretic mobility shift assays (EMSAs). The regulatory influence exerted by Pax6 on the alpha4 promoter was studied by transfections in RCECs. RESULTS: Expression of alpha4 was detected at both the mRNA and protein levels. Pax-6 was expressed in a cell-density-dependent manner in RCECs and altered the activity of the alpha4 promoter by interacting with multiple sites in both the promoter and 5'-flanking sequences. Pax-6 was also identified as the major protein component from the Bp5 complex, one of five protein complexes reported to bind the alpha4.1 element from the alpha4 basal promoter in vitro. CONCLUSIONS: These results provide evidence that the integrin subunit alpha4 and Pax-6 are coexpressed in RCECs and raise the possibility that Pax-6 directly regulates the expression of the alpha4 gene during corneal wound healing.

Regulation of the integrin subunit alpha5 gene promoter by the transcription factors Sp1/Sp3 is influenced by the cell density in rabbit corneal epithelial cells.

PURPOSE: Expression of the alpha5beta1 fibronectin (Fn) integrin is well recognized in the corneal epithelium and has been postulated to increase during wound healing. In the present study, the regulatory influence of the positive transcription factors Sp1/Sp3 on the activity directed by the promoter of the alpha5 gene was examined in rabbit corneal epithelial cells (RCECs) primary cultured at various cell densities. METHODS: Expression of the alpha5 subunit was assessed at the transcriptional level by semiquantitative RT-PCR analyses. The regulatory elements necessary to direct expression of the alpha5 gene were identified by transfecting RCECs with recombinant plasmids bearing various lengths from the alpha5 gene promoter fused to the CAT reporter gene. Binding of Sp1/Sp3 to the alpha5 promoter was assessed by both electrophoretic mobility shift assays (EMSAs) and DNaseI footprinting. Endogenous levels of Sp1/Sp3 were determined by Western blot and supershift analyses. The regulatory influence exerted by Sp1/Sp3 on the alpha5 promoter was evaluated both by site-directed mutagenesis and cotransfection in Sp1-deficient Drosophila SL-2 Schneider cells. RESULTS: Subconfluent RCECs expressed nearly five times more alpha5 transcript than 48-hour postconfluent RCECs. The activity directed by the alpha5 promoter was found to be affected by cell density. Strong promoter activity was observed in subconfluent RCECs, whereas a dramatic repression was measured in postconfluent cells. EMSA and DNaseI footprinting provided evidence for the binding of Sp1 to both a proximal site located within the previously reported alpha5 fibronectin responsive element (FRE), and a distal site located between positions -117 and -101. Cotransfection experiments in Schneider cells, as well as transfection of RCECs with recombinant constructs bearing mutations into the distal Sp1 site, confirmed the positive regulatory influence of Sp1 on both the -42/-92 and -92/-132 alpha5 promoter segments. Most of all, EMSA and Western blot analyses demonstrated the expression of substantial amounts of Sp1/Sp3 in subconfluent but not postconfluent RCECs. CONCLUSIONS: These results provide support to the hypothesis that the strong reduction in the activity of the alpha5 promoter when RCECs reach a high cell density is the consequence of a reduced expression of Sp1/Sp3 under such cell culture conditions.

Influence of sp1/sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues.

PURPOSE: Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS: HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS: Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS: Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes.