BACKGROUND
the glomerular filtration barrier consists of a fenestrated endothelium, a glomerular basement membrane and glomerular epithelial cells, podocytes.
podocytes surround the basement membrane of glomerular capillaries from the outside and present foot processes that are linked to each other with unique cell junction structures, the slit diaphragms .
according to the present view, sds form the final barrier preventing leakage of plasma proteins from circulation to urine  <cit> .
neph <dig>  also known as filtrin, is a member of the neph  family and shows sequence homology and structural similarity to two other neph proteins, neph <dig> and neph <dig>  and to nephrin  <cit> .
all these are transmembrane proteins that belong to the immunoglobulin superfamily  <cit> .
in podocytes, neph <dig>  like other neph family proteins and nephrin, localizes at the slit diaphragm  <cit> .
nephrin appears to be a key component of the sd and genetic nephrin deficiency results in the absence of sd and massive proteinuria in humans and mice  <cit> .
similarly, in neph1-deficient mice, the podocyte foot processes are effaced and the mice exhibit severe proteinuria  <cit> .
the function of neph <dig> in the kidney is less well known but sequence homology and similar location with other neph proteins and nephrin suggests that it has shared functions as a structural and signaling component of filtration barrier.
in addition, the expression of neph <dig> is down-regulated, similarly to nephrin mrna, in human proteinuric diseases proposing it to have a role in maintaining normal sd structure and function  <cit> .
however, very little is known about the mechanisms that regulate human neph genes and the mechanisms behind the transcriptional regulation of neph <dig> gene have not been elucidated at all.
to better understand the role of neph <dig> in the sd under normal and pathophysiological conditions, we investigated the transcriptional regulation of neph <dig> and identified the key regulatory regions in the neph <dig> 5' promoter.
further, we show that transcription factors nuclear factor-kappa b  and specificity protein  <dig>  bind to the promoter and are essential in controlling neph <dig> expression.
RESULTS
features of the upstream region of the human neph <dig> gene
the human neph <dig> gene  consists of fifteen exons.
it locates on chromosome 19q <dig> , adjacent to nephrin, and encodes a  <dig> kda protein.
there are at least  <dig> different splicing variants of neph <dig> that appear to have distinct tissue specificity  <cit> .
all known variants have the same transcription start site.
mouse and rat have syntenic neph <dig> gene regions in their chromosome locations 7qb <dig> and 1q <dig>  respectively.
we examined approximately  <dig> bp 5' flanking region upstream from the neph <dig> transcription start site .
neph <dig> proximal promoter near the transcription start site was noticed to lack a typical tata and caat boxes, but instead found to contain a highly gc-rich area.
a cpg island  was located around the proximal promoter from - <dig> to + <dig> using the blat tool .
cpg islands are generally found in the promoter regions of housekeeping genes and the cytosines within these regions are typically kept unmethylated to allow the expression of the correlated genes  <cit> .
the cpg island also includes several gc boxes, detected by matinspector software , that are important for the expression of many different ubiquitous as well as tissue-specific cellular and viral genes  <cit> .
two primate specific alu-repetition sequences were identified in the region - <dig> to - <dig> and from - <dig> to - <dig> bp upstream from the transcription start site using genomic repeats tool in genomatix.
location of the active promoter regions
to locate the minimal sequence responsible for the basal transcription of the human neph <dig> gene, a series of luciferase reporter plasmids containing various lengths of the neph <dig> 5'-flanking region were constructed and transiently transfected into human embryonic kidney cells  that express neph <dig> mrna endogenously .
as shown in figure  <dig>  already the neph <dig> 5' - <dig> promoter region activated the reporter gene to some extent, but the highest luciferase activity was produced by the neph <dig> 5' - <dig> construct.
this indicates that the essential regulatory elements necessary for the neph <dig> basal level transcription are located within the region of - <dig> to - <dig>  the constructs neph <dig> 5' - <dig>  neph <dig> 5' - <dig>  neph <dig> 5' - <dig>  neph <dig> 5' - <dig>  neph <dig> 5' - <dig> showed approximately the same luciferase activity.
neph <dig> 5' - <dig> construct and neph <dig> 5' - <dig> construct had significantly lower luciferase activities compared to luciferase activities of neph <dig> 5' - <dig> construct and neph <dig> 5' - <dig> construct, respectively, suggesting the presence of repressing transcription factor binding elements in the regions - <dig> to - <dig> and - <dig> to - <dig>  both identified alu repetition sequences locate in the region from - <dig> to - <dig> in the neph <dig> 5' - <dig> construct and may also be the reason for the lower activity of this part of the promoter.
the longest construct neph <dig> 5' - <dig> produced higher luciferase activity than neph <dig> 5' - <dig> or neph <dig> 5' - <dig> implying that in addition to proximal promoter region from - <dig> to - <dig>  there is possibly also another, weaker enhancer site in the distal 5' region from - <dig> to - <dig>
nf-κb and sp <dig> response elements are essential for the basal transcriptional activity of the neph <dig> promoter
reporter gene analysis indicated that the region of - <dig> to - <dig> in neph <dig> promoter contains the essential regulatory elements.
to locate putative binding sites for transcription factors within this promoter region, we used matinspector software.
the region was shown to contain putative binding sites for nf-κb  at the position - <dig> to - <dig> and for sp <dig>  at the position - <dig> to - <dig> .
core/matrix similarity values were  <dig> / <dig>  and  <dig> / <dig>  for the nf-κb and sp <dig> binding sites, respectively.
to investigate the functionality of these sites, nf-κb and sp <dig> elements were mutated in the neph <dig> 5' - <dig> construct and the constructs were transfected into a <dig> cells.
as shown in figure 3c, mutation of nf-κb element decreased transcriptional activity by 39% and mutation of sp <dig> element by even 80% indicating that both elements are functional and essential for the basal level transcription of neph <dig>  double mutation of both elements decreased transcriptional activity of neph <dig> 5' - <dig> region to the same level as sp <dig> binding site mutation alone.
overexpression of nf-κb and sp <dig> activate neph <dig> promoter
next we studied if nf-κb and sp <dig> are able to activate neph <dig> promoter.
nf-κb is a transcription factor family consisting of different subunits which function as homo- or heterodimers.
heterodimer consisting of p <dig> and p <dig> subunits and p <dig> homodimer are the well known dimers participating in gene transactivation  <cit> , and both p <dig> and p <dig> have been shown to be expressed in podocytes  <cit> .
therefore, we studied the effect of p <dig> and p <dig> overexpression on neph <dig> promoter activity.
neph <dig> 5' - <dig> construct was co-transfected together with nf-κb p <dig> and p <dig> expression vectors into a <dig> cells.
as shown in figure  <dig>  p <dig> over-expression resulted in activation of neph <dig> promoter with  <dig> -fold induction compared to control.
over-expression of p <dig> subunit had no effect on neph <dig> promoter activity.
simultaneous overexpression of p <dig> and p <dig> subunits led to no further activation of neph <dig> promoter, but instead, to lower levels of activity than with p <dig> subunit alone.
furthermore, luciferase activity of neph <dig> 5' - <dig> construct containing mutated nf-κb binding site was not altered.
these results suggested that nf-κb  is able to stimulate neph <dig> transcription through nf-κb binding site in the position of - <dig> to - <dig>
sp <dig> over-expression studies were carried out in drosophila schneider  <dig>  cells.
this is an established in vitro model to study the role of sp <dig>  since these cells lack endogenous sp <dig>  while all mammalian cell lines  have high endogenous level of sp <dig> and therefore are not suitable for sp <dig> over-expression studies  <cit> .
neph <dig> 5' - <dig> construct was co-transfected together with sp <dig> expression vector, and as shown in figure  <dig>  sp <dig> overexpression resulted in activation of neph <dig> promoter with  <dig> -fold induction compared to control.
in contrast, mutation of sp <dig> binding site abolished activation.
these results indicated that sp <dig> activates neph <dig> transcription through sp <dig> binding site in the position of - <dig> to - <dig>
overexpression of nf-κb increases neph <dig> mrna
we also examined whether nf-κb has an effect on the expression of endogenous neph <dig> mrna.
nf-κb p <dig> and p <dig> subunits were transiently transfected into a <dig> cells and after  <dig> h rna was extracted and neph <dig> mrna was quantified by quantitative pcr.
as shown in figure  <dig>  simultaneous over-expression of nf-κb p <dig> and p <dig> subunits increased neph <dig> mrna more than 4-fold compared to control.
a lesser, although significant, increase of neph <dig> mrna was also seen when p <dig> subunit was transfected alone.
in contrast, neph <dig> mrna level was not significantly different between p <dig> transfected and control cells.
these findings demonstrate that forced expression of nf-κb p <dig> and p <dig> drive neph <dig> gene expression in a <dig> cells.
nf-κb and sp <dig> bind to the promoter region of neph3
in order to study the interaction of nf-κb and sp <dig> with neph <dig> promoter ex vivo, a chromatin immunoprecipitation assay was carried out.
nf-κb p <dig>  p <dig> and sp <dig> antibodies were used to precipitate dna-protein complexes from conditionally immortalized human podocyte cell line that express neph <dig> mrna endogenously .
immunoprecipitated dna fragment including nf-κb and sp <dig> binding sites was amplified by real-time pcr.
both nf-κb subunit p <dig> and sp <dig> were shown to interact with neph <dig> promoter, whereas nf-κb p <dig> antibody and rabbit igg used as a negative control did not precipitate the promoter fragment .
due to different affinities of the used antibodies, the binding properties of the studied transcription factors cannot be compared.
discussion
neph <dig> is a member of the nephrin-like protein family and localizes at the slit diaphragms of podocyte foot processes which form the outermost layer of the kidney filtration barrier.
besides the sequence homology of neph <dig> with nephrin and other nephrin-like molecules, the known crucial components of the kidney filtration barrier, its structure, location, and down regulation in proteinuric diseases suggest that it plays an important role in maintaining the normal glomerular normal ultrafiltration.
the aim of this study was to gain insight into the transcriptional regulation of neph <dig> by identifying transcription factors that control neph <dig> expression.
using reporter gene constructs, we identified the neph <dig> 5' promoter region that is essential for the basal level transcription.
by computational analysis, the putative transcription factor binding elements for nf-κb and sp <dig> in this region were identified within this region and shown to be functional by site-directed mutagenesis.
furthermore, over-expression and chromatin immunoprecipitation studies indicated that nf-κb and sp <dig> indeed activate and bind to neph <dig> promoter.
sp <dig> is a member of sp family of transcription factors which contain a conserved set of cys2-his <dig> zinc-fingers representing a dna-binding domain.
sp <dig> is ubiquitously expressed in mammalian cells.
it can bind to and act through gc boxes to regulate gene expression of many housekeeping, tissue-specific, viral and inducible genes  <cit> .
previously, sp <dig> has been shown to play a role in regulating basal level transcription of two other podocyte proteins, cd2-associated protein  <cit>  and podocalyxin  <cit> .
sp <dig> is a constitutively active transcription factor  <cit> , and sp <dig> binding motifs are shown to be involved in the maintenance of the methylation-free status of the cpg islands  <cit> .
sp <dig> response element identified in this study also locates in the cpg island.
therefore, and similarly to the regulation of many other tata-less genes  <cit> , we propose that sp <dig> functions as a key regulator of the neph <dig> gene expression at the basal level.
nf-κb, a β-scaffold transcription factor family, plays an essential role in the regulation of many genes involved in immune and inflammatory responses, and in cell proliferation, differentiation and apoptosis  <cit> .
the subcellular location of nf-κb is controlled by interactions with inhibitory proteins of the iκb family which in response to a large variety of stimuli, such as stress and cytokines, are phosphorylated, ubiquitinated and degraded thereby freeing nf-κb to translocate to the nucleus and to bind dna at a response element of the consensus sequence 5'-gggrnnyycc-3'   <cit> .
nf-κb transcription factors exist as hetero- or homodimers and are formed by different subunits called p <dig>  p <dig>  c-rel, rela/p <dig> and relb.
heterodimer consisting of p <dig> and p <dig> subunits and p <dig> homodimer are the well known dimers and the most abundant ones in most cell types, including podocytes  <cit>  in our co-transfection experiments nf-κb p <dig> subunit together with neph <dig> reporter gene construct resulted in a significant increase in neph <dig> promoter activity.
similarly, the overexpression of p <dig> subunit increased endogenous neph <dig> mrna to some extend .
interestingly, overexpression of p <dig> and p <dig> subunits together resulted in a high increase in the neph <dig> mrna level, but the results from the reporter gene assay did not show further increase in promoter activation by combination of p <dig> and p <dig>  in addition, we could not detect p <dig> binding to the promoter in the chip assay.
it might be that in the chromosomal context of the endogenous gene the p <dig> subunit binds the promoter and activates the neph <dig> expression together with p <dig> although this is not seen with reporter gene constructs or fragmented chromosomes used in chip.
however, based on these results it is difficult to conclude whether it is p <dig> homodimer or p50/p <dig> heterodimer that activates the transcription of the neph <dig>  most likely, different nf-κb subunits may heterodimerize with p <dig> and regulate the expression of neph <dig> in different ways under various physiological conditions.
we have previously shown that neph <dig> mrna is decreased in the kidneys of the diabetic nephropathy patients  <cit> .
in line with this, nephrin mrna has been shown to be downregulated in human diabetic nephropathy  <cit>  and in addition, during the development of diabetic nephropathy in a rat model, nephrin mrna is first transiently increased and later down-regulated  <cit> .
interestingly, the activation and nuclear translocation of nf-κb subunits p <dig> and p <dig> have been demonstrated in human diabetic nephropathy  <cit> .
this, together with our results showing that nf-κb regulates neph <dig> expression at the basal level , prompts for further studies investigating whether nf-κb plays a role in the regulation of neph <dig>  under diabetic conditions.
the studies with renal biopsies from patients with acquired human glomerular diseases show correlation, besides in the mrna expression of neph <dig> and nephrin  <cit> , also in the mrna expression of nephrin and other podocyte-expressed genes, including synaptopodin and α-actinin- <dig>  <cit> .
this suggests that common regulatory mechanisms may be activated in proteinuric glomerular diseases.
neph <dig> locates on the same chromosome as nephrin, 19q <dig> , where these genes are arranged in a head-to-head orientation separated by a 5-kb region  <cit> .
the genomic arrangement of neph <dig> and nephrin together with their similar structure and location, in addition to the correlation in the mrna expression of various podocyte-expressed genes  <cit>  proposes that neph <dig> and other podocyte genes may share key features in their regulation.
however, whether nf-κb and sp <dig> control the expression of nephrin or other podocyte genes is yet to be clarified.
besides nf-κb and sp <dig>  additional transcription factors, both activating and inhibiting, are likely to participate in the regulation of neph <dig>  it has been shown that at least transcription factor wilms tumor- <dig>  snail, retinoic acid receptors and peroxisome proliferator-activated receptors regulate nephrin  <cit> , and could be involved also in neph <dig> regulation.
interestingly, sp <dig> has been demonstrated to interact with nf-κb, and cooperative functional and physical interactions between sp <dig> and nf-κb have been shown to be required for example, for hiv- <dig> enhancer activation  <cit> .
our co-trasfection assays with sp <dig> and nf-κb in a <dig> cells didn't show any activation of the neph <dig> promoter co-operatively between sp <dig> and nf-κb .
however, sp <dig> overexpression studies are not feasible in mammalian cell lines because of their high endogenous level of sp <dig>  which may be near saturating with regard to induction of reporter gene expression.
regardless, it is possible that nf-κb and sp <dig> function cooperatively in the neph <dig> gene regulation at the endogenous level.
in addition to kidney, neph <dig> is also expressed in other tissues including the β-cells of the pancreas  <cit>  and in the developing central nervous system in the brain where it is transiently expressed in early postmitotic neural precursor cells  <cit> .
it is suggested that in pancreas and in brain neph <dig> is involved in maintaining cell-cell contacts  <cit> , but the exact function of neph <dig> in these tissues has not yet been elucidated.
different splicing variants of neph <dig> also appear to have distinct tissue specificity.
whether nf-κb and sp <dig> also regulate neph <dig> expression and the production of its different splicing variants in non-renal tissues remains to be elucidated.
CONCLUSIONS
in conclusion, in this study we show that the main activating regulatory region of the neph <dig> gene locates in its proximal promoter region between nucleotides - <dig> and - <dig>  further, we show that transcription factors sp <dig> and nf-κb bind to this promoter region and are involved in the transcriptional regulation of the neph <dig> gene at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of neph <dig> gene.

 the glomerular filtration barrier consists of a fenestrated endothelium, a glomerular basement membrane and glomerular epithelial cells, podocytes. podocytes surround the basement membrane of glomerular capillaries from the outside and present foot processes that are linked to each other with unique cell junction structures, the slit diaphragms. according to the present view, sds form the final barrier preventing leakage of plasma proteins from circulation to urine. neph also known as filtrin, is a member of the neph family and shows sequence homology and structural similarity to two other neph proteins, neph and neph and to nephrin. all these are transmembrane proteins that belong to the immunoglobulin superfamily. in podocytes, neph like other neph family proteins and nephrin, localizes at the slit diaphragm. nephrin appears to be a key component of the sd and genetic nephrin deficiency results in the absence of sd and massive proteinuria in humans and mice. similarly, in neph1-deficient mice, the podocyte foot processes are effaced and the mice exhibit severe proteinuria. the function of neph in the kidney is less well known but sequence homology and similar location with other neph proteins and nephrin suggests that it has shared functions as a structural and signaling component of filtration barrier. in addition, the expression of neph is down-regulated, similarly to nephrin mrna, in human proteinuric diseases proposing it to have a role in maintaining normal sd structure and function. however, very little is known about the mechanisms that regulate human neph genes and the mechanisms behind the transcriptional regulation of neph gene have not been elucidated at all. to better understand the role of neph in the sd under normal and pathophysiological conditions, we investigated the transcriptional regulation of neph and identified the key regulatory regions in the neph 5' promoter. further, we show that transcription factors nuclear factor-kappa b and specificity protein bind to the promoter and are essential in controlling neph expression.  features of the upstream region of the human neph gene the human neph gene consists of fifteen exons. it locates on chromosome 19q, adjacent to nephrin, and encodes a kda protein. there are at least different splicing variants of neph that appear to have distinct tissue specificity. all known variants have the same transcription start site. mouse and rat have syntenic neph gene regions in their chromosome locations 7qb and 1q respectively. we examined approximately bp 5' flanking region upstream from the neph transcription start site. neph proximal promoter near the transcription start site was noticed to lack a typical tata and caat boxes, but instead found to contain a highly gc-rich area. a cpg island was located around the proximal promoter from - to + using the blat tool. cpg islands are generally found in the promoter regions of housekeeping genes and the cytosines within these regions are typically kept unmethylated to allow the expression of the correlated genes. the cpg island also includes several gc boxes, detected by matinspector software, that are important for the expression of many different ubiquitous as well as tissue-specific cellular and viral genes. two primate specific alu-repetition sequences were identified in the region - to - and from - to - bp upstream from the transcription start site using genomic repeats tool in genomatix. location of the active promoter regions to locate the minimal sequence responsible for the basal transcription of the human neph gene, a series of luciferase reporter plasmids containing various lengths of the neph 5'-flanking region were constructed and transiently transfected into human embryonic kidney cells that express neph mrna endogenously. as shown in figure already the neph 5' - promoter region activated the reporter gene to some extent, but the highest luciferase activity was produced by the neph 5' - construct. this indicates that the essential regulatory elements necessary for the neph basal level transcription are located within the region of - to - the constructs neph 5' - neph 5' - neph 5' - neph 5' - neph 5' - showed approximately the same luciferase activity. neph 5' - construct and neph 5' - construct had significantly lower luciferase activities compared to luciferase activities of neph 5' - construct and neph 5' - construct, respectively, suggesting the presence of repressing transcription factor binding elements in the regions - to - and - to - both identified alu repetition sequences locate in the region from - to - in the neph 5' - construct and may also be the reason for the lower activity of this part of the promoter. the longest construct neph 5' - produced higher luciferase activity than neph 5' - or neph 5' - implying that in addition to proximal promoter region from - to - there is possibly also another, weaker enhancer site in the distal 5' region from - to - nf-κb and sp response elements are essential for the basal transcriptional activity of the neph promoter reporter gene analysis indicated that the region of - to - in neph promoter contains the essential regulatory elements. to locate putative binding sites for transcription factors within this promoter region, we used matinspector software. the region was shown to contain putative binding sites for nf-κb at the position - to - and for sp at the position - to -. core/matrix similarity values were / and / for the nf-κb and sp binding sites, respectively. to investigate the functionality of these sites, nf-κb and sp elements were mutated in the neph 5' - construct and the constructs were transfected into a cells. as shown in figure 3c, mutation of nf-κb element decreased transcriptional activity by 39% and mutation of sp element by even 80% indicating that both elements are functional and essential for the basal level transcription of neph double mutation of both elements decreased transcriptional activity of neph 5' - region to the same level as sp binding site mutation alone. overexpression of nf-κb and sp activate neph promoter next we studied if nf-κb and sp are able to activate neph promoter. nf-κb is a transcription factor family consisting of different subunits which function as homo- or heterodimers. heterodimer consisting of p and p subunits and p homodimer are the well known dimers participating in gene transactivation, and both p and p have been shown to be expressed in podocytes. therefore, we studied the effect of p and p overexpression on neph promoter activity. neph 5' - construct was co-transfected together with nf-κb p and p expression vectors into a cells. as shown in figure p over-expression resulted in activation of neph promoter with -fold induction compared to control. over-expression of p subunit had no effect on neph promoter activity. simultaneous overexpression of p and p subunits led to no further activation of neph promoter, but instead, to lower levels of activity than with p subunit alone. furthermore, luciferase activity of neph 5' - construct containing mutated nf-κb binding site was not altered. these results suggested that nf-κb is able to stimulate neph transcription through nf-κb binding site in the position of - to - sp over-expression studies were carried out in drosophila schneider cells. this is an established in vitro model to study the role of sp since these cells lack endogenous sp while all mammalian cell lines have high endogenous level of sp and therefore are not suitable for sp over-expression studies. neph 5' - construct was co-transfected together with sp expression vector, and as shown in figure sp overexpression resulted in activation of neph promoter with -fold induction compared to control. in contrast, mutation of sp binding site abolished activation. these results indicated that sp activates neph transcription through sp binding site in the position of - to - overexpression of nf-κb increases neph mrna we also examined whether nf-κb has an effect on the expression of endogenous neph mrna. nf-κb p and p subunits were transiently transfected into a cells and after h rna was extracted and neph mrna was quantified by quantitative pcr. as shown in figure simultaneous over-expression of nf-κb p and p subunits increased neph mrna more than 4-fold compared to control. a lesser, although significant, increase of neph mrna was also seen when p subunit was transfected alone. in contrast, neph mrna level was not significantly different between p transfected and control cells. these findings demonstrate that forced expression of nf-κb p and p drive neph gene expression in a cells. nf-κb and sp bind to the promoter region of neph3 in order to study the interaction of nf-κb and sp with neph promoter ex vivo, a chromatin immunoprecipitation assay was carried out. nf-κb p p and sp antibodies were used to precipitate dna-protein complexes from conditionally immortalized human podocyte cell line that express neph mrna endogenously. immunoprecipitated dna fragment including nf-κb and sp binding sites was amplified by real-time pcr. both nf-κb subunit p and sp were shown to interact with neph promoter, whereas nf-κb p antibody and rabbit igg used as a negative control did not precipitate the promoter fragment. due to different affinities of the used antibodies, the binding properties of the studied transcription factors cannot be compared. discussion neph is a member of the nephrin-like protein family and localizes at the slit diaphragms of podocyte foot processes which form the outermost layer of the kidney filtration barrier. besides the sequence homology of neph with nephrin and other nephrin-like molecules, the known crucial components of the kidney filtration barrier, its structure, location, and down regulation in proteinuric diseases suggest that it plays an important role in maintaining the normal glomerular normal ultrafiltration. the aim of this study was to gain insight into the transcriptional regulation of neph by identifying transcription factors that control neph expression. using reporter gene constructs, we identified the neph 5' promoter region that is essential for the basal level transcription. by computational analysis, the putative transcription factor binding elements for nf-κb and sp in this region were identified within this region and shown to be functional by site-directed mutagenesis. furthermore, over-expression and chromatin immunoprecipitation studies indicated that nf-κb and sp indeed activate and bind to neph promoter. sp is a member of sp family of transcription factors which contain a conserved set of cys2-his zinc-fingers representing a dna-binding domain. sp is ubiquitously expressed in mammalian cells. it can bind to and act through gc boxes to regulate gene expression of many housekeeping, tissue-specific, viral and inducible genes. previously, sp has been shown to play a role in regulating basal level transcription of two other podocyte proteins, cd2-associated protein and podocalyxin. sp is a constitutively active transcription factor, and sp binding motifs are shown to be involved in the maintenance of the methylation-free status of the cpg islands. sp response element identified in this study also locates in the cpg island. therefore, and similarly to the regulation of many other tata-less genes, we propose that sp functions as a key regulator of the neph gene expression at the basal level. nf-κb, a β-scaffold transcription factor family, plays an essential role in the regulation of many genes involved in immune and inflammatory responses, and in cell proliferation, differentiation and apoptosis. the subcellular location of nf-κb is controlled by interactions with inhibitory proteins of the iκb family which in response to a large variety of stimuli, such as stress and cytokines, are phosphorylated, ubiquitinated and degraded thereby freeing nf-κb to translocate to the nucleus and to bind dna at a response element of the consensus sequence 5'-gggrnnyycc-3'. nf-κb transcription factors exist as hetero- or homodimers and are formed by different subunits called p p c-rel, rela/p and relb. heterodimer consisting of p and p subunits and p homodimer are the well known dimers and the most abundant ones in most cell types, including podocytes in our co-transfection experiments nf-κb p subunit together with neph reporter gene construct resulted in a significant increase in neph promoter activity. similarly, the overexpression of p subunit increased endogenous neph mrna to some extend. interestingly, overexpression of p and p subunits together resulted in a high increase in the neph mrna level, but the results from the reporter gene assay did not show further increase in promoter activation by combination of p and p in addition, we could not detect p binding to the promoter in the chip assay. it might be that in the chromosomal context of the endogenous gene the p subunit binds the promoter and activates the neph expression together with p although this is not seen with reporter gene constructs or fragmented chromosomes used in chip. however, based on these results it is difficult to conclude whether it is p homodimer or p50/p heterodimer that activates the transcription of the neph most likely, different nf-κb subunits may heterodimerize with p and regulate the expression of neph in different ways under various physiological conditions. we have previously shown that neph mrna is decreased in the kidneys of the diabetic nephropathy patients. in line with this, nephrin mrna has been shown to be downregulated in human diabetic nephropathy and in addition, during the development of diabetic nephropathy in a rat model, nephrin mrna is first transiently increased and later down-regulated. interestingly, the activation and nuclear translocation of nf-κb subunits p and p have been demonstrated in human diabetic nephropathy. this, together with our results showing that nf-κb regulates neph expression at the basal level, prompts for further studies investigating whether nf-κb plays a role in the regulation of neph under diabetic conditions. the studies with renal biopsies from patients with acquired human glomerular diseases show correlation, besides in the mrna expression of neph and nephrin, also in the mrna expression of nephrin and other podocyte-expressed genes, including synaptopodin and α-actinin-. this suggests that common regulatory mechanisms may be activated in proteinuric glomerular diseases. neph locates on the same chromosome as nephrin, 19q, where these genes are arranged in a head-to-head orientation separated by a 5-kb region. the genomic arrangement of neph and nephrin together with their similar structure and location, in addition to the correlation in the mrna expression of various podocyte-expressed genes proposes that neph and other podocyte genes may share key features in their regulation. however, whether nf-κb and sp control the expression of nephrin or other podocyte genes is yet to be clarified. besides nf-κb and sp additional transcription factors, both activating and inhibiting, are likely to participate in the regulation of neph it has been shown that at least transcription factor wilms tumor- snail, retinoic acid receptors and peroxisome proliferator-activated receptors regulate nephrin, and could be involved also in neph regulation. interestingly, sp has been demonstrated to interact with nf-κb, and cooperative functional and physical interactions between sp and nf-κb have been shown to be required for example, for hiv- enhancer activation. our co-trasfection assays with sp and nf-κb in a cells didn't show any activation of the neph promoter co-operatively between sp and nf-κb. however, sp overexpression studies are not feasible in mammalian cell lines because of their high endogenous level of sp which may be near saturating with regard to induction of reporter gene expression. regardless, it is possible that nf-κb and sp function cooperatively in the neph gene regulation at the endogenous level. in addition to kidney, neph is also expressed in other tissues including the β-cells of the pancreas and in the developing central nervous system in the brain where it is transiently expressed in early postmitotic neural precursor cells. it is suggested that in pancreas and in brain neph is involved in maintaining cell-cell contacts, but the exact function of neph in these tissues has not yet been elucidated. different splicing variants of neph also appear to have distinct tissue specificity. whether nf-κb and sp also regulate neph expression and the production of its different splicing variants in non-renal tissues remains to be elucidated.  in conclusion, in this study we show that the main activating regulatory region of the neph gene locates in its proximal promoter region between nucleotides - and - further, we show that transcription factors sp and nf-κb bind to this promoter region and are involved in the transcriptional regulation of the neph gene at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of neph gene. 
using promoter reporter gene constructs, we localized the main activating regulatory region of neph gene in its proximal promoter region from - to - within this region, putative transcription factor binding sites for nf-κb and sp were found by computational analysis. our results show that nf-κb and sp are key regulators of neph expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of neph gene. neph is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. the exact function of neph in the kidney is not known but we have previously shown that the level of neph mrna is decreased in proteinuric diseases. neph protein shows homology and structural similarity to neph neph and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. mutational screening indicated that nf-κb and sp response elements are essential for the basal transcriptional activity of the neph promoter. in this study we investigated the transcriptional regulation of neph gene by identifying transcription factors that control neph expression. neph proximal promoter near the transcription start site was found to be devoid of tata and caat boxes, but to contain a highly gc-rich area. chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both nf-κb and sp interact with the neph promoter.
 neph is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. neph protein shows homology and structural similarity to neph neph and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. the exact function of neph in the kidney is not known but we have previously shown that the level of neph mrna is decreased in proteinuric diseases. this suggests that neph may play a role in the pathogenesis of kidney damage, and emphasizes the need to analyze the regulatory mechanisms of neph gene. in this study we investigated the transcriptional regulation of neph gene by identifying transcription factors that control neph expression.  we cloned and characterized approximately kb fragment upstream of the neph gene. neph proximal promoter near the transcription start site was found to be devoid of tata and caat boxes, but to contain a highly gc-rich area. using promoter reporter gene constructs, we localized the main activating regulatory region of neph gene in its proximal promoter region from - to - within this region, putative transcription factor binding sites for nf-κb and sp were found by computational analysis. mutational screening indicated that nf-κb and sp response elements are essential for the basal transcriptional activity of the neph promoter. co-transfection studies further showed that nf-κb and sp regulate neph promoter activity. in addition, overexpression of nf-κb increased endogenous neph gene expression. chromatin immunoprecipitation assay using cultured human podocytes demonstrated that both nf-κb and sp interact with the neph promoter.  our results show that nf-κb and sp are key regulators of neph expression at the basal level in podocytes, therefore providing new insight into the molecular mechanisms that contribute to the expression of neph gene. 