BACKGROUND
fibin, a secreted protein, was recently described as a novel growth factor found in various embryonic tissues of zebrafish and in the forelimb buds of mouse embryos  <cit> .
the fibin sequence exhibits no homology to any known proteins.
in zebrafish, the functional knockdown of fibin resulted in an absence of pectoral fins  <cit> .
there are a number of factors involved in limb development; among them retinaldehyde dehydrogenase type  <dig>  <cit> , the fibroblast growth factors fgf <dig>  <cit> , fgf <dig>  <cit>  and the transcription factor tbx <dig>  <cit> .
mutations in the tbx <dig> gene cause the holt-oram syndrome, which is characterized by defects of the upper limbs and heart  <cit> .
interestingly, fibin induces the expression of tbx <dig> via an unknown signalling cascade  <cit> .
the findings suggest that fibin is essential for the pectoral fin bud formation during embryogenesis.
however, human hereditary diseases caused by mutation of the fibin gene are not known yet.
fibin attracted our attention because fibin mrna levels were increased in the kidneys of newborn mice with x-linked nephrogenic diabetes insipidus  <cit> .
further, in-depth screening of public sources revealed that fibin appears to be highly expressed not only in embryonic stages, but also in a number of tissues in postnatal and adult stages, which implicates functions after embryonic ontogenesis.
in the present study, we provide comprehensive data on its evolutionary origin, structural conservation, expression profiles, expression regulation, cellular targeting and protein biochemistry which includes heterologous protein expression and refolding.
although fibin is routed through the endoplasmic reticulum  we found no significant evidence of secretion.
we show that fibin is posttranslationally modified and forms dimers when heterologously expressed.
our data suggest that fibin may require yet unknown accessory proteins for proper function.
RESULTS
genomic organization, expression and expression regulation of fibin
initial investigations mining public sequence data sources  revealed that human and mouse fibin transcripts are present in a number of different tissues, such as bones, kidney and lung, and also during several ontogenetic stages .
comparison of fibin mrna and genomic sequences in humans and mice provided no indication of transcript splicing or editing.
more detailed information of the genomic structure and transcript length are shown in additional file  <dig>  figure s <dig>  to map putative cis-acting elements in the 5' flanking region the matinspector online software was used.
we identified several binding sites for the glucocorticoid receptor, camp responsive element protein, nf-κb and wnt-activated lef/tcf transcriptional factors .
to experimentally substantiate est data, we performed qpcr studies with cdna of various tissues and at different mouse embryonic stages in relation to β2-microglobulin.
as shown in figure 2a, increased fibin expression was found in the cerebellum and skeletal muscle.
high expression levels of fibin mrna were not only present in limbs of the mice at days  <dig> and  <dig> but also in the trunk .
overall fibin expression was higher in fetal than adult tissues.
next, we screened several human and mouse cell lines for possible fibin expression.
significant mrna levels were found in hek <dig>  ht <dig>  hepg <dig>  and l <dig> cells , whereas t98g and raw <dig>  cells presented very low fibin mrna levels.
other human , mouse  and rat  cell lines showed no detectable fibin mrna .
to analyze fibin expression regulation, l <dig> and hek <dig> cells were incubated with various compounds known to have effects on the expression of other genes.
as shown in figure 3a, manganese chloride significantly increased fibin expression in l <dig> cells.
the highest levels were obtained after  <dig> hours.
dexamethasone and phorbol ester also significantly increased fibin expression but to a lesser extent than manganese chloride .
in hek <dig> cells , none of the compounds tested had any effect on the fibin mrna level.
all attempts to quantify endogenous fibin expression in embryonic and adult tissues and cell lines at the protein level failed so far, probably because the protein amounts were below western blot sensitivity .
further, specific detection after enrichment of fibin from tissue  and from cell line  extracts by immunoprecipitation and immunoaffinity chromatography followed by western blotting was unsuccessful.
both antibodies, against the whole protein and c terminus, have a detection limit of approximately  <dig> fmol fibin per lane  in western blots indicating that amounts of fibin from tissue and cell extracts were below this concentration.
posttranslational proteolytic processing, as cause for the lack of immunodetection, can almost be excluded because posttranslational cleavage sites of prohormone convertases  were cloned into the firefly luciferase gene containing the pgl <dig> reporter vector .
the transcriptional start is located between - <dig> bp and - <dig> bp upstream of the translational start and there is no intron in the 5' utr .
maximum promoter activities in both, l <dig> and hek <dig> cells were found with constructs containing - <dig> bp upstream of the genomic sequence  indicating similar regulation of basal transcription in both cell lines.
mouse raw <dig>  cells and human t98g cells served as the respective negative controls because no endogenous fibin mrna expression was found in either cell lines .
to analyze whether the  <dig> -kbp-promoter construct contains regulatory sites, transfected l <dig> and hek <dig> cells were incubated with dexamethasone, phorbol ester and manganese chloride.
as shown in figure  <dig>  significant regulation of luciferase activities by these three compounds was found in l <dig> cells but not in hek <dig> cells.
subcellular distribution of fibin
to study the subcellular distribution of fibin a c-terminally gfp-tagged version was heterologously expressed in cos- <dig> cells.
as demonstrated in figure 6a fibin-gfp transfected cells displayed a reticular fluorescence pattern suggesting association of fibin with the er.
co-immunostaining with an antibody against the er-specific protein calnexin showed co-localization .
only a very low signal overlap was seen with golgi-specific protein gm <dig> .
an identical subcellular distribution was found for wild-type fibin using the mouse fibin-specific antibody .
because no structural signatures of transmembrane domains were predicted we speculate that the n terminus contains a signal sequence guiding the protein to the er.
to address this hypothesis we generated an n-terminally truncated fibin-gfp construct starting with the methionine at position  <dig>  as shown in figure 6e this construct was homogenously distributed in the cell indicating that the deleted n terminus contained a signal sequence for er localization.
to finally verify whether the putative er-targeting signal is cleaved from the precursor protein we purified a fibin construct c-terminal tagged with his <dig> from cos- <dig> cells under denaturating conditions and sequenced the n terminus by the edman method .
the native n terminus started with the amino acids tyr-phe-asp, which correspond to the amino acid positions  <dig> to  <dig> in fibin.
this indicates that fibin contains an n-terminal signal sequence of  <dig> amino acids which is removed by cleaving between gly <dig> and tyr <dig>
posttranslational modifications and molecular forms of fibin
to further substantiate the molecular architecture of fibin, protein isolation and western blot studies were performed.
ortholog alignment and analysis of the fibin amino acid sequence by prediction software revealed an n-terminal signal sequence for er entry and a conserved n-glycosylation site .
the theoretical molecular weights of murine non-glycosylated full length fibin and fibin without the signal sequence were calculated as  <dig>  kda and  <dig>  kda, respectively.
as demonstrated in figure 7a, fibin appeared at  <dig> kda in both the soluble and membranous fractions of transfected cos- <dig> cells.
furthermore, we studied fibin glycosylation, a clear marker for er/golgi passage.
deglycosylation with pngase f and mutation  of the potential n-glycosylation site at amino acid position  <dig> led to the calculated size of approximate  <dig> kda .
fibin heterologously expressed in e. coli was used as a control as it was the same size as the two deglycosylated forms .
fibin contains two conserved cys  residues .
mutagenesis studies together with sds-page/western blot analyses under non-reducing and reducing conditions were performed to test whether these cys residues participate in intra- and/or intermolecular disulfide bonds.
under non-reducing conditions, specific bands were found most prominently at  <dig> and  <dig> kda, which correspond to the monomeric and homodimeric forms of fibin, respectively .
the dimeric form completely disappeared under reducing conditions.
none of the individual cys mutations  promoted the monomeric form.
only the double mutant cys52ser/cys64ser resulted in a complete loss of dimer formation .
these findings support fibin as a dimer where two molecules are linked via two disulfide bonds between cys52-cys <dig> and cys64-cys <dig>  in the case of only one  or random disulfide bonds one would expect an increase of the monomeric form at least in one of the cys mutants.
to test whether fibin also exists in non-covalent multimeric forms, size exclusion chromatography experiments were performed.
protein from fibin-myc-transfected cos- <dig> cells was released from microsomes by french press treatment into hypotonic buffer.
the extract was applied to a superdex  <dig> column and analyzed by size exclusion chromatography.
fractions were separated by sds-page under non-reducing conditions and analyzed by a western blot.
as shown in additional file  <dig>  figure s <dig>  a reasonable amount of sds-resistant fibin multimers were found in high molecular weight fractions .
these bands probably represent soluble aggregates.
fibin dimers and monomers were mainly eluted in fractions below  <dig> kda.
fibin is not secreted from cos- <dig> cells
to further address the fate of fibin, serum-free medium supernatants of transfected cos- <dig> cells were collected, concentrated by trichloroacetic acid precipitation, and subjected to western blot analysis.
first, we analyzed the release of fibin, c-myc-tagged version of fibin, the secretory alkaline phosphatase  and gfp  after  <dig> h incubation with serum-free medium.
additionally, transfected cells were treated with brefeldin a , an inhibitor of golgi function that blocks the classical secretory pathway  <cit> .
as shown in figure  <dig> seap was only found in the medium but not in cell lysates.
the secretion of seap was completely blocked by bfa .
fibin and fibin-myc were detected in the cell lysates and medium.
the release of fibin and fibin-myc was not blocked by bfa.
gapdh, an endogenous cytosolic protein, and the heterologously expressed, cytosolic gfp were also found in the cell lysates and medium.
these results suggested that fibin found in the medium was not actively secreted but rather passively released after cell death.
to minimize unspecific release by cell damage the incubation time was reduced to  <dig> hours and a lower bfa concentration  was used .
seap was found in the supernatant whereas fibin was only found in the cell lysate.
discussion
fibin is a structurally and evolutionary preserved vertebrate protein with no amino acid homology to known proteins .
previous studies demonstrated the functional relevance of fibin during embryonic development of pectoral fin bud initiation in zebrafish and limb development in mice  <cit> .
indeed, fibin mrna is embryonically expressed not only in forelimbs, but also in other tissues.
furthermore, we found reasonable fibin expression in adult tissues, with the highest expression levels in cerebellum and skeletal muscle; however, fibin mrna was also present in most other tissues investigated .
these data are consistent with the abundance of fibin est in many different tissues including tumors and embryonic stages in mouse and humans indicating a role for fibin during both ontogenesis and adolescence.
recent microarray-based gene expression studies revealed an increase in fibin expression within a right cardiac ventricle during chronic pulmonary embolism  <cit> .
this may suggest an involvement of fibin in myocardial pathogenic or remodelling processes.
the fibin gene displays a simple genomic structure without introns and a transcription start in close proximity to the translational start, aug. transcription regulation was studied in cell lines constitutively expressing fibin.
several compounds known to activate transcription factors which potentially bind in the promoter region of fibin  were tested.
lithium chloride activates the transcription factors lef/tcf of the wnt/β-catenin pathway via blocking gsk3β  <cit> .
dexamethasone, a glucocorticoid, binds to intracellular receptors which act as transcription factor.
forskolin increases camp via adenylyl cyclase and camp activates protein kinase a and the transcription factor creb.
manganese ions can mediate effects via the nf-κb/ap- <dig> signalling  <cit>  and the nrf2/are cascade  <cit> .
phorbol ester activates the protein kinase c/map kinase pathway.
in l <dig> cells, but not hek <dig> cells, manganese ions, phorbol ester and dexamethasone induced fibin-mrna expression with highest mrna levels 8- <dig> h after stimulation .
the genomic sequence ~ <dig> bp upstream of the transcriptional start is sufficient to mediate basal transcription in hek <dig> and l <dig> cells, but not in human and mouse cell lines without endogenous fibin expression .
interestingly, and in contrast to l <dig> cells, hek <dig> cells lack factor which are required for transcription regulation upon stimulation with manganese ions, phorbol ester and dexamethasone .
previous studies showed that fibin expression in vivo depends on the presence of wnt2b, indicating regulation of fibin expression by the wnt/β-catenin pathway  <cit> .
indeed, there is a lef-1/tcf target sequence upstream of the fibin coding sequence .
deletion of this sequence in our  <dig> -kbp-reporter construct had no specific effect on basal luciferase activity in l <dig> or hek <dig> cells .
no effect on luciferase activity was found when constitutively active catenin was co-expressed with the  <dig> -kbp-fibin promoter construct in hek <dig> and l <dig> cells.
using the tcf reporter gene kit  as control the wnt/β-catenin pathway was active in hek <dig> but not in l <dig> cells .
fibin expression was not increased in l <dig> and hek <dig> cells when transfected with constitutively active β-catenin .
moreover, lithium chloride, known to activate wnt/β-catenin pathway, had no effect on fibin expression .
these results indicate that the wnt/β-catenin pathway is probably not involved in fibin regulation at least in the investigated cell line.
according to our in silico analysis, which predicted a signal sequence for er entry and a glycosylation site, we experimentally demonstrated fibin localization in the er  and cleavage of signal sequence and glycosylation at asn <dig> .
sds-page under non-reducing conditions and immunoblotting revealed mostly multimeric fibin formed by an intermolecular disulfide bond between the two cysteine residues, cys <dig> and cys <dig>  .
it remains unclear whether homodi- and homomultimers are the natural forms of fibin.
we approached the quarternary structure of fibin by size exclusion chromatography.
we found almost all the fibin in non-covalent associated multimeric complexes .
when expressed in e. coli, fibin was exclusively found in inclusion bodies.
refolding with various protocols and under different reducing conditions led to a protein with high aggregation tendency .
expression of a thioredoxin-fibin fusion protein in e. coli origami b  resulted in lower inclusion body formation, but the soluble protein also formed multimeric complexes when analyzed by size exclusion chromatography .
the high tendency of fibin to form multimeric complexes seems to be similar to other secretory proteins, such as adiponectin  <cit> .
although fibin displays properties of a secretory protein , no experimental evidence for substantial fibin secretion, beyond fibin release through cell death, was found in cos- <dig> cells .
this finding is in contrast to a previous study  <cit>  which, however, lacked controls for unspecific release.
our data suggest at least three scenarios: first, fibin naturally exists as a monomer or as a homodimer formed by disulfide bridges.
the insufficient secretion found in our heterologous cos- <dig> cell overexpression system may result from the naturally high aggregation tendency of fibin, which prevents proper trafficking to the golgi complex.
in a second scenario, fibin needs escort proteins for secretion which are not expressed in cos- <dig> cells.
third, fibin forms heteromers with a yet unknown protein.
the lack of the partner protein would prevent proper folding and trafficking, and explains the aggregation and retention tendency of fibin in er.
there are a number of examples of the latter scenario in wnt ligand family members  <cit> , interleukin  <dig>   <cit> , and cbln <dig>  <cit> .
at present, all attempts failed to prove or to reject one of these hypotheses.
yeast two-hybrid experiments using human fibin and a human kidney library revealed expected interaction partners, such as chaperones and the exocyst complex component  <dig>  but no putative fibin subunits .
until endogenous fibin is purified from natural sources, clarification of its functional molecular structure will remain unknown.
CONCLUSIONS
fibin appears to play a role not only during embryonic development but also in many adult functions.
fibin expression shows complex regulation via the signalling pathways of protein kinase c, glucocorticoids and manganese ion-activated transcriptional factors.
although fibin displays all features of a secretory protein, proper secretion presumably requires a currently unknown covalently linked or associated cofactor.
because fibin is evolutionarily old and lacks structurally related paralogs, it may have a unique functionality; conditional gene deficiency in mouse models may provide further insights into its function during different ontogenetic stages and in different tissues.

 fibin, a secreted protein, was recently described as a novel growth factor found in various embryonic tissues of zebrafish and in the forelimb buds of mouse embryos. the fibin sequence exhibits no homology to any known proteins. in zebrafish, the functional knockdown of fibin resulted in an absence of pectoral fins. there are a number of factors involved in limb development; among them retinaldehyde dehydrogenase type, the fibroblast growth factors fgf, fgf and the transcription factor tbx. mutations in the tbx gene cause the holt-oram syndrome, which is characterized by defects of the upper limbs and heart. interestingly, fibin induces the expression of tbx via an unknown signalling cascade. the findings suggest that fibin is essential for the pectoral fin bud formation during embryogenesis. however, human hereditary diseases caused by mutation of the fibin gene are not known yet. fibin attracted our attention because fibin mrna levels were increased in the kidneys of newborn mice with x-linked nephrogenic diabetes insipidus. further, in-depth screening of public sources revealed that fibin appears to be highly expressed not only in embryonic stages, but also in a number of tissues in postnatal and adult stages, which implicates functions after embryonic ontogenesis. in the present study, we provide comprehensive data on its evolutionary origin, structural conservation, expression profiles, expression regulation, cellular targeting and protein biochemistry which includes heterologous protein expression and refolding. although fibin is routed through the endoplasmic reticulum we found no significant evidence of secretion. we show that fibin is posttranslationally modified and forms dimers when heterologously expressed. our data suggest that fibin may require yet unknown accessory proteins for proper function.  genomic organization, expression and expression regulation of fibin initial investigations mining public sequence data sources revealed that human and mouse fibin transcripts are present in a number of different tissues, such as bones, kidney and lung, and also during several ontogenetic stages. comparison of fibin mrna and genomic sequences in humans and mice provided no indication of transcript splicing or editing. more detailed information of the genomic structure and transcript length are shown in additional file figure s to map putative cis-acting elements in the 5' flanking region the matinspector online software was used. we identified several binding sites for the glucocorticoid receptor, camp responsive element protein, nf-κb and wnt-activated lef/tcf transcriptional factors. to experimentally substantiate est data, we performed qpcr studies with cdna of various tissues and at different mouse embryonic stages in relation to β2-microglobulin. as shown in figure 2a, increased fibin expression was found in the cerebellum and skeletal muscle. high expression levels of fibin mrna were not only present in limbs of the mice at days and but also in the trunk. overall fibin expression was higher in fetal than adult tissues. next, we screened several human and mouse cell lines for possible fibin expression. significant mrna levels were found in hek ht hepg and l cells, whereas t98g and raw cells presented very low fibin mrna levels. other human, mouse and rat cell lines showed no detectable fibin mrna. to analyze fibin expression regulation, l and hek cells were incubated with various compounds known to have effects on the expression of other genes. as shown in figure 3a, manganese chloride significantly increased fibin expression in l cells. the highest levels were obtained after hours. dexamethasone and phorbol ester also significantly increased fibin expression but to a lesser extent than manganese chloride. in hek cells, none of the compounds tested had any effect on the fibin mrna level. all attempts to quantify endogenous fibin expression in embryonic and adult tissues and cell lines at the protein level failed so far, probably because the protein amounts were below western blot sensitivity. further, specific detection after enrichment of fibin from tissue and from cell line extracts by immunoprecipitation and immunoaffinity chromatography followed by western blotting was unsuccessful. both antibodies, against the whole protein and c terminus, have a detection limit of approximately fmol fibin per lane in western blots indicating that amounts of fibin from tissue and cell extracts were below this concentration. posttranslational proteolytic processing, as cause for the lack of immunodetection, can almost be excluded because posttranslational cleavage sites of prohormone convertases were cloned into the firefly luciferase gene containing the pgl reporter vector. the transcriptional start is located between - bp and - bp upstream of the translational start and there is no intron in the 5' utr. maximum promoter activities in both, l and hek cells were found with constructs containing - bp upstream of the genomic sequence indicating similar regulation of basal transcription in both cell lines. mouse raw cells and human t98g cells served as the respective negative controls because no endogenous fibin mrna expression was found in either cell lines. to analyze whether the -kbp-promoter construct contains regulatory sites, transfected l and hek cells were incubated with dexamethasone, phorbol ester and manganese chloride. as shown in figure significant regulation of luciferase activities by these three compounds was found in l cells but not in hek cells. subcellular distribution of fibin to study the subcellular distribution of fibin a c-terminally gfp-tagged version was heterologously expressed in cos- cells. as demonstrated in figure 6a fibin-gfp transfected cells displayed a reticular fluorescence pattern suggesting association of fibin with the er. co-immunostaining with an antibody against the er-specific protein calnexin showed co-localization. only a very low signal overlap was seen with golgi-specific protein gm. an identical subcellular distribution was found for wild-type fibin using the mouse fibin-specific antibody. because no structural signatures of transmembrane domains were predicted we speculate that the n terminus contains a signal sequence guiding the protein to the er. to address this hypothesis we generated an n-terminally truncated fibin-gfp construct starting with the methionine at position as shown in figure 6e this construct was homogenously distributed in the cell indicating that the deleted n terminus contained a signal sequence for er localization. to finally verify whether the putative er-targeting signal is cleaved from the precursor protein we purified a fibin construct c-terminal tagged with his from cos- cells under denaturating conditions and sequenced the n terminus by the edman method. the native n terminus started with the amino acids tyr-phe-asp, which correspond to the amino acid positions to in fibin. this indicates that fibin contains an n-terminal signal sequence of amino acids which is removed by cleaving between gly and tyr posttranslational modifications and molecular forms of fibin to further substantiate the molecular architecture of fibin, protein isolation and western blot studies were performed. ortholog alignment and analysis of the fibin amino acid sequence by prediction software revealed an n-terminal signal sequence for er entry and a conserved n-glycosylation site. the theoretical molecular weights of murine non-glycosylated full length fibin and fibin without the signal sequence were calculated as kda and kda, respectively. as demonstrated in figure 7a, fibin appeared at kda in both the soluble and membranous fractions of transfected cos- cells. furthermore, we studied fibin glycosylation, a clear marker for er/golgi passage. deglycosylation with pngase f and mutation of the potential n-glycosylation site at amino acid position led to the calculated size of approximate kda. fibin heterologously expressed in e. coli was used as a control as it was the same size as the two deglycosylated forms. fibin contains two conserved cys residues. mutagenesis studies together with sds-page/western blot analyses under non-reducing and reducing conditions were performed to test whether these cys residues participate in intra- and/or intermolecular disulfide bonds. under non-reducing conditions, specific bands were found most prominently at and kda, which correspond to the monomeric and homodimeric forms of fibin, respectively. the dimeric form completely disappeared under reducing conditions. none of the individual cys mutations promoted the monomeric form. only the double mutant cys52ser/cys64ser resulted in a complete loss of dimer formation. these findings support fibin as a dimer where two molecules are linked via two disulfide bonds between cys52-cys and cys64-cys in the case of only one or random disulfide bonds one would expect an increase of the monomeric form at least in one of the cys mutants. to test whether fibin also exists in non-covalent multimeric forms, size exclusion chromatography experiments were performed. protein from fibin-myc-transfected cos- cells was released from microsomes by french press treatment into hypotonic buffer. the extract was applied to a superdex column and analyzed by size exclusion chromatography. fractions were separated by sds-page under non-reducing conditions and analyzed by a western blot. as shown in additional file figure s a reasonable amount of sds-resistant fibin multimers were found in high molecular weight fractions. these bands probably represent soluble aggregates. fibin dimers and monomers were mainly eluted in fractions below kda. fibin is not secreted from cos- cells to further address the fate of fibin, serum-free medium supernatants of transfected cos- cells were collected, concentrated by trichloroacetic acid precipitation, and subjected to western blot analysis. first, we analyzed the release of fibin, c-myc-tagged version of fibin, the secretory alkaline phosphatase and gfp after h incubation with serum-free medium. additionally, transfected cells were treated with brefeldin a, an inhibitor of golgi function that blocks the classical secretory pathway. as shown in figure seap was only found in the medium but not in cell lysates. the secretion of seap was completely blocked by bfa. fibin and fibin-myc were detected in the cell lysates and medium. the release of fibin and fibin-myc was not blocked by bfa. gapdh, an endogenous cytosolic protein, and the heterologously expressed, cytosolic gfp were also found in the cell lysates and medium. these results suggested that fibin found in the medium was not actively secreted but rather passively released after cell death. to minimize unspecific release by cell damage the incubation time was reduced to hours and a lower bfa concentration was used. seap was found in the supernatant whereas fibin was only found in the cell lysate. discussion fibin is a structurally and evolutionary preserved vertebrate protein with no amino acid homology to known proteins. previous studies demonstrated the functional relevance of fibin during embryonic development of pectoral fin bud initiation in zebrafish and limb development in mice. indeed, fibin mrna is embryonically expressed not only in forelimbs, but also in other tissues. furthermore, we found reasonable fibin expression in adult tissues, with the highest expression levels in cerebellum and skeletal muscle; however, fibin mrna was also present in most other tissues investigated. these data are consistent with the abundance of fibin est in many different tissues including tumors and embryonic stages in mouse and humans indicating a role for fibin during both ontogenesis and adolescence. recent microarray-based gene expression studies revealed an increase in fibin expression within a right cardiac ventricle during chronic pulmonary embolism. this may suggest an involvement of fibin in myocardial pathogenic or remodelling processes. the fibin gene displays a simple genomic structure without introns and a transcription start in close proximity to the translational start, aug. transcription regulation was studied in cell lines constitutively expressing fibin. several compounds known to activate transcription factors which potentially bind in the promoter region of fibin were tested. lithium chloride activates the transcription factors lef/tcf of the wnt/β-catenin pathway via blocking gsk3β. dexamethasone, a glucocorticoid, binds to intracellular receptors which act as transcription factor. forskolin increases camp via adenylyl cyclase and camp activates protein kinase a and the transcription factor creb. manganese ions can mediate effects via the nf-κb/ap- signalling and the nrf2/are cascade. phorbol ester activates the protein kinase c/map kinase pathway. in l cells, but not hek cells, manganese ions, phorbol ester and dexamethasone induced fibin-mrna expression with highest mrna levels 8- h after stimulation. the genomic sequence ~ bp upstream of the transcriptional start is sufficient to mediate basal transcription in hek and l cells, but not in human and mouse cell lines without endogenous fibin expression. interestingly, and in contrast to l cells, hek cells lack factor which are required for transcription regulation upon stimulation with manganese ions, phorbol ester and dexamethasone. previous studies showed that fibin expression in vivo depends on the presence of wnt2b, indicating regulation of fibin expression by the wnt/β-catenin pathway. indeed, there is a lef-1/tcf target sequence upstream of the fibin coding sequence. deletion of this sequence in our -kbp-reporter construct had no specific effect on basal luciferase activity in l or hek cells. no effect on luciferase activity was found when constitutively active catenin was co-expressed with the -kbp-fibin promoter construct in hek and l cells. using the tcf reporter gene kit as control the wnt/β-catenin pathway was active in hek but not in l cells. fibin expression was not increased in l and hek cells when transfected with constitutively active β-catenin. moreover, lithium chloride, known to activate wnt/β-catenin pathway, had no effect on fibin expression. these results indicate that the wnt/β-catenin pathway is probably not involved in fibin regulation at least in the investigated cell line. according to our in silico analysis, which predicted a signal sequence for er entry and a glycosylation site, we experimentally demonstrated fibin localization in the er and cleavage of signal sequence and glycosylation at asn. sds-page under non-reducing conditions and immunoblotting revealed mostly multimeric fibin formed by an intermolecular disulfide bond between the two cysteine residues, cys and cys. it remains unclear whether homodi- and homomultimers are the natural forms of fibin. we approached the quarternary structure of fibin by size exclusion chromatography. we found almost all the fibin in non-covalent associated multimeric complexes. when expressed in e. coli, fibin was exclusively found in inclusion bodies. refolding with various protocols and under different reducing conditions led to a protein with high aggregation tendency. expression of a thioredoxin-fibin fusion protein in e. coli origami b resulted in lower inclusion body formation, but the soluble protein also formed multimeric complexes when analyzed by size exclusion chromatography. the high tendency of fibin to form multimeric complexes seems to be similar to other secretory proteins, such as adiponectin. although fibin displays properties of a secretory protein, no experimental evidence for substantial fibin secretion, beyond fibin release through cell death, was found in cos- cells. this finding is in contrast to a previous study which, however, lacked controls for unspecific release. our data suggest at least three scenarios: first, fibin naturally exists as a monomer or as a homodimer formed by disulfide bridges. the insufficient secretion found in our heterologous cos- cell overexpression system may result from the naturally high aggregation tendency of fibin, which prevents proper trafficking to the golgi complex. in a second scenario, fibin needs escort proteins for secretion which are not expressed in cos- cells. third, fibin forms heteromers with a yet unknown protein. the lack of the partner protein would prevent proper folding and trafficking, and explains the aggregation and retention tendency of fibin in er. there are a number of examples of the latter scenario in wnt ligand family members, interleukin, and cbln. at present, all attempts failed to prove or to reject one of these hypotheses. yeast two-hybrid experiments using human fibin and a human kidney library revealed expected interaction partners, such as chaperones and the exocyst complex component but no putative fibin subunits. until endogenous fibin is purified from natural sources, clarification of its functional molecular structure will remain unknown.  fibin appears to play a role not only during embryonic development but also in many adult functions. fibin expression shows complex regulation via the signalling pathways of protein kinase c, glucocorticoids and manganese ion-activated transcriptional factors. although fibin displays all features of a secretory protein, proper secretion presumably requires a currently unknown covalently linked or associated cofactor. because fibin is evolutionarily old and lacks structurally related paralogs, it may have a unique functionality; conditional gene deficiency in mouse models may provide further insights into its function during different ontogenetic stages and in different tissues. 
although fibin displays all features of a secretory protein, it is mostly retained in the endoplasmic reticulum when heterologously expressed. fibin was initially discovered as a secreted signal molecule essential for pectoral fin bud initiation in zebrafish. fibin is expressed in cerebellum, skeletal muscle and many other embryonic and adult mouse tissues suggesting not only a role during embryonic development but also in adult functions. a -kbp genomic sequence fragment upstream of the coding sequence is sufficient to drive and regulate fibin expression through stimulation by glucocorticoids, activators of the protein kinase c signalling pathways and manganese ions. however, proper secretion presumably requires an unknown covalently-linked or associated co-factor. currently, there is little information about the molecular architecture and biological relevance of fibin in humans and other mammals. fibin is an evolutionarily conserved protein, carries a cleavable signal peptide and is glycosylated at asn the two conserved cysteines participate in intermolecular disulfide bond and multimer formation.
 fibin was initially discovered as a secreted signal molecule essential for pectoral fin bud initiation in zebrafish. currently, there is little information about the molecular architecture and biological relevance of fibin in humans and other mammals.  fibin is expressed in cerebellum, skeletal muscle and many other embryonic and adult mouse tissues suggesting not only a role during embryonic development but also in adult functions. a -kbp genomic sequence fragment upstream of the coding sequence is sufficient to drive and regulate fibin expression through stimulation by glucocorticoids, activators of the protein kinase c signalling pathways and manganese ions. fibin is an evolutionarily conserved protein, carries a cleavable signal peptide and is glycosylated at asn the two conserved cysteines participate in intermolecular disulfide bond and multimer formation. although fibin displays all features of a secretory protein, it is mostly retained in the endoplasmic reticulum when heterologously expressed.  fibin is functionally relevant during embryogenesis and adult life. its expression is regulated by a number of cellular signalling pathways and the protein is routed via the secretory pathway. however, proper secretion presumably requires an unknown covalently-linked or associated co-factor. 