BACKGROUND
members of the erbb receptor tyrosine kinase family  and their ligands are important regulators of cell growth and differentiation.
studies of erbb <dig>  erbb <dig> and heregulin  deficient mice indicate that these genes are essential for embryonic development  <cit> .
in turn, the activity of the erbb receptors is regulated by their interacting partners.
an erbb <dig> binding protein  was cloned in our laboratory during a yeast two-hybrid screen  <cit> .
ebp <dig> is identical to the murine p38-2g <dig> protein which was isolated as a dna binding protein <cit> .
these proteins are members of the proliferation-associated 2g <dig>  gene family, which is highly conserved throughout evolution  <cit> .
more than  <dig> genes encoding proteins homologous to ebp <dig> have been found in organisms ranging from danio rerio to pan troglodytes  <cit> .
ebp <dig> is expressed in mammalian cell lines derived from multiple origins.
ebp <dig> mrna is also found in all normal adult human and murine tissues examined including liver, heart, brain, placenta, lung, muscle, pancreas, kidney, prostate and breast  <cit> .
examination of est data bases reveals that ebp <dig> is expressed in all tissue types at different stages of embryonic development.
its ubiquitous distribution in both embryonic and adult tissues suggests it affects various developmental and physiological pathways  <cit> .
an important role for ebp <dig> in cell proliferation and survival in vitro has been demonstrated by many groups.
we have shown that ebp <dig> inhibits transcription of e2f <dig> regulated cell cycle genes such as e2f <dig>  cyclin d <dig> and cyclin e  <cit> .
this transcriptional regulation is due in part to interactions with the transcriptional corepressors sin3a, rb and hdac <dig> on e2f <dig> regulated promoters  <cit> .
ectopic expression of ebp <dig> inhibits the growth of human breast <cit>  and prostate cancer cells  <cit>  and fibroblasts  <cit>  both in vitro and in animal models  <cit> .
in breast cancer cell lines, ebp <dig> regulates levels of erbb <dig> and controls the cellular response to heregulin and the antiestrogen tamoxifen  <cit> .
in prostate cancer, ectopic expression of ebp <dig> results in downregulation of androgen receptor  and several of its target genes and inhibition of ar-regulated cell growth  <cit> .
ebp <dig> also has a role in regulating cell survival as its interaction with akt kinase suppresses apoptosis <cit> .
the mechanisms by which ebp <dig> exerts its effects on cell proliferation and survival are incompletely understood.
the biological effects of ebp <dig> were originally postulated to be based on its ability to bind dna.
ebp <dig> is a member of the sf <dig> protein superfamily, the prototype of which is a  <dig> kda dna binding protein isolated from the fission yeast s. pombe  <cit> .
blast analysis reveals that ebp <dig> and the yeast  <dig> kda protein have 38% amino acid identity and 56% similarity.
the s. pombe protein preferentially binds a synthetic curved dna sequence.
ebp <dig> binds directly to this synthetic curved dna sequence and is recruited to e2f <dig> promoter elements in vitro and in vivo as part of a protein complex  <cit> .
the interaction of ebp <dig> with the e2f <dig> promoter is regulated by the erbb <dig> ligand hrg.
in addition to its ability to interact with proteins and dna, ebp <dig> binds to an array of rna targets.
squaritto et al  <cit>  found that a pool of ebp <dig> localized to the nucleolus binds rna and may be involved in ribosomal biogenesis.
ebp <dig> can bind to b <dig> , as a component of pre- ribosomal ribonucleoprotein complexes  <cit> .
in the cytoplasm, ebp <dig> associates with the  <dig> s subunit of mature ribosomes, suggesting that it can also be involved in protein translation.
sedimentation studies revealed that ebp <dig> copurified with eif2α, a component of the translation initiation complex  <cit> .
ebp <dig> contains an rna binding domain σ-  <dig> like motif that maps to aa 46– <dig> that mediates its interaction with rna and its nucleolar localization  <cit> .
a double-stranded rna binding domain was also mapped to aa 91– <dig>  other studies showing that ebp <dig> binds the 3'utr of bcl- <dig> mrna and stabilizes β-globin-are bcl- <dig> transcripts  <cit>  suggest that ebp <dig> is a functional rna binding protein.
finally, ebp <dig> has been demonstrated to bind to viral internal ribosome entry sites   <cit> .
despite burgeoning interest in the role of ebp <dig> in several important cellular activities, the physiological function of ebp <dig> in the intact organism is not known.
we therefore characterized ebp1- deficient mice generated by a gene trap insertion in intron  <dig> of the pa2g <dig> gene.
we show that ebp1-deficient mice exhibit transient dwarfism.
this is accompanied by a decrease in serum levels of igf- <dig> and igf binding proteins and changes in expression of several other genes associated with cell growth.
these results provide in vivo evidence that ebp <dig> plays an important role in cellular growth.
RESULTS
disruption of the ebp <dig> gene
ebp1+/- mice were generated at lexicon genetics from es cells containing a retroviral gene trap in the ebp <dig> locus as described previously  <cit> .
sequence analysis showed that the gene trap was inserted at a single site in the  <dig>  kb intron  <dig> of the ebp <dig> gene .
the insertion led to the expression of an ebp1-neomycin fusion molecule containing only the first  <dig> amino acids that are encoded by exon  <dig> of ebp <dig> that has no known biological function.
the mice were genotyped by pcr with primers located at each side of the gene trap and in the ltr <dig> cassette .
the absence of ebp <dig> mrna in ebp1-/- mice was confirmed by rt-pcr with β actin as an internal control on total mrna from  <dig>  day embryo fibroblasts.
the transcript was present in fibroblasts from wild type, but not ebp1-/- mef .
in addition, western blot analysis indicated that ebp <dig> protein was not expressed in knock out mefs .
ebp <dig> expression in adult life
examination of est data bases and our own published data  <cit>  indicate that ebp <dig> is ubiquitously expressed in normal adult mouse tissue.
we therefore measured ebp <dig> protein expression in several organs to determine the penetrance of the knock out.
we found as expected that ebp <dig> was expressed in adult lung and kidney of  <dig> week old wild type mice.
ebp <dig> expression was absent in the ebp1-/- mouse .
breeding data and post natal lethality
breeding data were obtained from crosses between both heterozygous and homozygous mice, since both ebp1+/- and ebp1-/-male and females were fertile.
in all cases, the sex ratio was approximately 1: <dig>  the genotypes of the pups from heterozygous pairs were present at approximately the expected ratio of 25:50: <dig>  .
survival was approximately equal for all three groups eight weeks after birth.
male and female knock-out mice were fertile and inbred mating resulted in viable offspring.
however, litter size was smaller for the ebp1-/- mice.
litter size of wild type mice was  <dig>  ± .
<dig>  and for knock out mice, <dig>  ±  <dig>  .
in general, life expectancy, of knock out mice was indistinguishable compared with their littermates.
ten mice of each genotype were maintained for one year.
9/ <dig> knock out and 10/ <dig> heterozygous and 10/ <dig> wild type mice survived.
reduced growth of ebp1-/- mice
the most striking feature of adult ebp1-/- mice is their initial smaller body size  and in about 1/ <dig> of the mice the presence of kinky tails.
the progeny of crosses between heterozygous parents were kept with the heterozygous mother and weighed sequentially.
at day  <dig>  there was a significant  reduction of approximately 30% in body weight for both males and females.
mice were weaned at day  <dig>  after day  <dig> mice began to catch up in size with wild type litter mates and at day  <dig> there were no differences in weight .
to rule out the possibility that competition during milk sucking was responsible for growth retardation, ebp1-/- males and females or wild type males and females were crossed.
ko or wt progeny were housed alone with their mother.
males and females were weighed and data pooled due to difficulties in determining sex at early time points.
weights were the same for wt and ko mice at day  <dig>  however, at days  <dig> and  <dig>  these mice also displayed a 25% reduction in weight .
macro and microscopic appearance of organs
to identify development defects and or pathological features, we examined the macro and microscopic appearance of a series of organs from adult ebp <dig> mice.
there were no gross morphological differences between the ebp1+/+ and ebp1-/- mice at  <dig> weeks of age.
the examination of hematoxylin and eosin stained sections from organs revealed no histological changes in gut, kidney, lymph nodes, testes, liver, spleen, pancreas, skeletal muscle, lung, testis and ovaries.
peripheral blood counts and blood chemistries  were within normal limits.
glucose levels were  <dig> ±  <dig> mg/dl for knock-out mice and  <dig> ±  <dig> mg/dl for wild type mice.
proliferative behavior of mefs
as ebp1-/- mice were small and the ebp <dig> gene was originally identified as a proliferation associated gene, we examined the proliferative behavior of  <dig>  day mouse embryo fibroblasts.
under the standard culture conditions used, the number of mefs from ebp1-/- mice was lower at all the time points tested .
in addition, we were able to propagate wild type mefs for  <dig> passages, but ebp1-/- mefs for only  <dig>  a senescent morphology was observed at passage  <dig> for knock out mefs .
target gene and global expression profiling in mefs
to obtain additional information about the cellular pathways involved in the phenotype of the mefs, we performed global expression profiling of matched  <dig>  day mefs.
the expression data have been deposited with the geo data base  as gse <dig>  a total of  <dig> genes were changed more than two fold .
among the transcripts involved in growth regulation, we found that several members of the insulin, egfr, cyclin and mapk families were significantly changed .
to verify these findings, we first examined the expression of cyclin d <dig> which has previously been shown to be repressed by ectopic expression of ebp <dig> in human breast cancer cell lines  <cit> .
we found by rt-pcr and quantitative pcr analysis that cyclin d <dig> mrna was increased .
however, cyclin d <dig> protein levels were equal in wild type and knock out mefs reflecting the complex regulation of cyclin d <dig> protein expression  <cit>  .
three regulators of the insulin signaling pathway were also found to be significantly decreased.
these include igf- <dig>  igfbp- <dig> and igfbp- <dig>  the levels of igf- <dig> were measured by elisa in conditioned media derived from wild type and knock out mefs.
the concentration of igf- <dig> in conditioned media from  <dig> day cultures of mefs was  <dig> ±  <dig> pg/ml for knock out cells and  <dig> ±  <dig> pg/ml for wild type mefs.
alterations of egf-regulated signaling pathways
our microarray data indicated that two members of the mapk signal transduction pathway, sos <dig> and grb <dig> were upregulated in knock-out as compared with wild type mefs.
we verified by western blot analysis that sos was increased in ebp <dig> knock out mefs as compared with wild type cells .
we therefore tested if the cellular response to egf would be changed in ebp <dig> knock out mefs.
murine mefs are known to express egfr and egfr mrna expression was not changed in ebp <dig> knock outs  <cit> .
cells were treated with egf  for the times indicated and mapk activation assessed by detection of phosphorylated mapk by western blot analysis.
the results indicated that both wild type and knock out mefs could respond to egf stimulation as evidenced by phosphorylation of mapk.
however, mapk was basally phosphorylated only in knock out mefs .
egf-induced akt phosphorylation in wild type and knock out cells was also determined using a phospho specific akt antibody.
the response to egf was equivalent in wild type and knock out cells .
changes in expression of proliferation associated genes in adult target tissues
previous in vitro work with ebp <dig> has used breast and prostate cancer cell lines derived from adults.
in these tissues, the expression of cyclin d <dig> <cit> , agr <dig>  <cit>  and androgen receptor  <cit>  have been shown to be regulated by ebp <dig>  we therefore examined expression of these genes in adult mammary and prostate epithelium.
we found that in mammary epithelial cells, cyclin d <dig> protein levels were not changed in knock out mice as compared to wild type .
however, the phosphorylation of akt was greatly increased in ebp1-/- mammary tissue .
in prostate cancer cell lines, the expression of the androgen receptor and the metastasis associated protein agr <dig> has been demonstrated to be decreased by ectopic expression of ebp <dig>  we found that agr <dig> mrna  and protein  expression and ar protein expression  were increased in prostates of ebp <dig> knock out mice as compared to age matched controls.
finally, based on data from mefs, we examined serum levels of igf- <dig> from age matched wild type and knock out mice by elisa assays.
igf- <dig> was reduced to 66% of wild type levels in serum of  <dig> month old mice .
in addition, western blot analysis of adult liver and kidney  indicated that igfbp- <dig> was reduced in knock-out versus wild type organs .
discussion
to characterize the physiological role of ebp <dig>  we generated ebp <dig> deficient mice exhibiting a gene trap introduced into intron  <dig> of the ebp <dig> gene.
the gene trap leads to deletion of all but the first  <dig> amino acids of ebp <dig> which have no biological function on their own.
the most striking feature of the ebp1-/- phenotype was the small size of the mice.
it is of interest to note that inhibition of expression of the potato homologue of ebp <dig> also results in a dwarf phenotype  <cit> .
ebp1-/- pups were on average 25% smaller than their wild type and heterozygous littermates until day  <dig>  the approximate onset of puberty.
mice were well proportioned with no skeletal abnormalities other than a kinky tail in approximately 1/ <dig> of the mice.
an unusual feature of our in vivo analysis is the fact that the reduced weight was recovered after the prepubescent stage, while ebp <dig> is still normally transcribed and translated and therefore likely to have a regulatory role.
although we are unable to explain the mechanism underlying this recovery, we report this observation as a possible example of temporal restricted competence in growth regulation of specific cell types by ebp <dig>  this phenotype is similar to that observed for the otx  <dig> gene, a homeobox containing gene that in vitro activates transcription of gh, fsh, and lh  <cit> .
it is of interest that the phenotype of ebp <dig> knock out animals also resembles that of the imp <dig> knock out mice  <cit> .
imp <dig> belongs to a family of rna binding proteins implicated in mrna localization, turnover, and translational control.
these mice also exhibit a dwarf phenotype with reduced proliferation of knock out mefs.
in these mice, growth retardation is related to a hypoplastic small intestine.
in contrast, ebp <dig> mice exhibit a transient dwarfism and their intestines are both grossly and histologically normal at  <dig> weeks.
however, we were unable to assess intestinal anatomy and histology at earlier time points, and it remains possible that abnormalities occur when the weight differences are most marked.
ebp <dig> has also been shown to be an rna binding protein with a sigma-  <dig> like motif.
this motif has been identified as a new rna binding domain unique to the superfamily of imp <dig> or brix proteins that are required for ribosome biogenesis  <cit> .
a role for ebp <dig> in ribosomal biogenesis is supported by studies indicating its localization in the nucleolus, its association with  <dig> s ribosomal rna, and its complexing with proteins such as b <dig> which are involved in ribosome processing <cit> .
whereas the significance of rna binding proteins in spatial and temporal control of gene expression during development is evident in invertebrate organisms, the function of rna binding proteins during mammalian development is less clear.
with the exception of fmrp, dazla, and nova- <dig>  which are associated with mental retardation, azospermia, and neuronal viability respectively  <cit> , the majority of the mammalian rna binding proteins are constitutively expressed and exhibit a relative broad rna-binding specificity.
further work is needed to uncover the role that ebp1's rna binding activity has in the development of this phenotype and the target rnas that specifically interact with ebp <dig>
the molecular pathways leading to dwarfism are likely to involve several mechanisms.
as reduced growth was manifested in the mefs from day  <dig>  embryos, we used these cells to measure changes in gene expression associated with loss of ebp <dig>  global gene expression analysis indicated that expression of igf- <dig> and the igf binding proteins igfbp <dig> and  <dig> was decreased.
the in vivo growth promoting role of the igfs has been demonstrated conclusively from the dwarfing phenotypes observed after targeted mutagenesis of the mouse igf <dig> and igf1r genes.
<cit> .
igf- <dig> functions both during embryonic and post natal development.
igf- <dig> mutants exhibit a birth weight that is 60% of normal and the difference between their size and that of wild type mice increases progressively with age  <cit> .
the ebp <dig> phenotype is not as severe as that observed in igf- <dig> deficient mice.
the function of igfbps in cell growth is more complex.
however, one role is to sequester and stabilize igf- <dig>  <cit> .
thus, decreases in igfbps may result in destabilization of igf- <dig> and may be partially responsible for lower levels of serum igf- <dig>
we found that mapk was constitutively phosphorylated in mefs derived from knock out mice.
this constitutive phosphorylation was associated with the overexpression of sos- <dig>  an upstream activator of the ras -mapk signal transduction pathway.
it is possible that overexpression of sos- <dig> led to increased basal phosphorylation of mapk.
in contrast, both the basal and hrg-induced activation of the mapk pathway is increased in ebp <dig> transfected breast cancer cell lines  <cit> .
the reasons for these apparently discordant findings may be related to the ligand used, the differential expression of erbb receptors in breast cancer cells versus fibroblasts, and the cellular background.
we have previously shown that inhibition of ebp <dig> expression results in increases in phospho akt in human breast cancer cell lines, whereas ectopic expression of ebp <dig> inhibits hrg induced akt activation  <cit> .
here, we found that ebp <dig> had no effect on akt phosphorylation after egf stimulation of mefs.
however, akt was constitutively phosphorylated in mammary epithelial cells of knock-out mice.
the mechanism of the increase in akt phosphorylation is unclear.
paradoxically, ahn et al  <cit>  have shown the binding of ebp <dig> to nuclear akt increases akt phosphorylation  <cit>  in pc- <dig> neuronal cells.
it is not known if ebp <dig> binds akt in normal mammary epithelial cells or affects its phosphorylation.
these studies point up the importance of tissue context in the activation of specific signaling molecules.
as activation of akt is important in development of mammary adenocarcinoma  <cit> , we are currently monitoring breast tumor incidence with age in ebp <dig> knock out mice.
we have not yet seen increased tumor incidence in a small cohort of mice that have been carried until one year.
finally, our laboratory has demonstrated that ectopic expression of ebp <dig> results in decreased expression of ar and ar target genes and inhibition of growth of prostate cancer cells both in vivo and in vitro  <cit> .
in the current study, deletion of the ebp <dig> gene led to increased expression of ar in prostates of adult mice and upregulation of the metastasis associated gene agr <dig>  ar plays a key role in the development and progression of prostate cancer, and we anticipate that the ebp <dig> knockout mouse may have a hyperactivated ar signaling axis that may result in increased incidence of prostate cancer.
also with regard to prostate cancer, decreased expression of igfbp- <dig> is correlated with increased risk of developing prostate cancer <cit> .
western blot analysis verified that ebp <dig> knockout led to reduced protein levels of igfbp- <dig> in liver and kidney.
we were unable to detect igfbp- <dig> in prostates of either wild type or knock out mice, but studies are ongoing to assess if there are changes in igfbp- <dig> expression in the prostates of knock out mice.
we are also monitoring the incidence of prostate cancer of the ebp1-/- mice as they age.
nevertheless, current studies, together with our previous reports, strongly suggest that ebp <dig>  as an endogenous regulator of erbb-ar crosstalk, may regulate expression of a series of genes that are involved in aggressive prostate cancer growth.
CONCLUSIONS
in summary, although ebp <dig> appears to be dispensable for prenatal development, loss of ebp <dig> affects post-natal growth.
this growth delay may be related to changes in igf- <dig> and igfbp levels and to changes in key components of cellular proliferation pathways.
of interest, two genes involved in prostate cancer progression, androgen receptor and agr <dig>  are increased in prostates of ebp <dig> knock out mice.
our ebp1-/- mouse line represents a new in vivo model to investigate ebp <dig> function in the entire organism.

 members of the erbb receptor tyrosine kinase family and their ligands are important regulators of cell growth and differentiation. studies of erbb erbb and heregulin deficient mice indicate that these genes are essential for embryonic development. in turn, the activity of the erbb receptors is regulated by their interacting partners. an erbb binding protein was cloned in our laboratory during a yeast two-hybrid screen. ebp is identical to the murine p38-2g protein which was isolated as a dna binding protein. these proteins are members of the proliferation-associated 2g gene family, which is highly conserved throughout evolution. more than genes encoding proteins homologous to ebp have been found in organisms ranging from danio rerio to pan troglodytes. ebp is expressed in mammalian cell lines derived from multiple origins. ebp mrna is also found in all normal adult human and murine tissues examined including liver, heart, brain, placenta, lung, muscle, pancreas, kidney, prostate and breast. examination of est data bases reveals that ebp is expressed in all tissue types at different stages of embryonic development. its ubiquitous distribution in both embryonic and adult tissues suggests it affects various developmental and physiological pathways. an important role for ebp in cell proliferation and survival in vitro has been demonstrated by many groups. we have shown that ebp inhibits transcription of e2f regulated cell cycle genes such as e2f cyclin d and cyclin e. this transcriptional regulation is due in part to interactions with the transcriptional corepressors sin3a, rb and hdac on e2f regulated promoters. ectopic expression of ebp inhibits the growth of human breast and prostate cancer cells and fibroblasts both in vitro and in animal models. in breast cancer cell lines, ebp regulates levels of erbb and controls the cellular response to heregulin and the antiestrogen tamoxifen. in prostate cancer, ectopic expression of ebp results in downregulation of androgen receptor and several of its target genes and inhibition of ar-regulated cell growth. ebp also has a role in regulating cell survival as its interaction with akt kinase suppresses apoptosis. the mechanisms by which ebp exerts its effects on cell proliferation and survival are incompletely understood. the biological effects of ebp were originally postulated to be based on its ability to bind dna. ebp is a member of the sf protein superfamily, the prototype of which is a kda dna binding protein isolated from the fission yeast s. pombe. blast analysis reveals that ebp and the yeast kda protein have 38% amino acid identity and 56% similarity. the s. pombe protein preferentially binds a synthetic curved dna sequence. ebp binds directly to this synthetic curved dna sequence and is recruited to e2f promoter elements in vitro and in vivo as part of a protein complex. the interaction of ebp with the e2f promoter is regulated by the erbb ligand hrg. in addition to its ability to interact with proteins and dna, ebp binds to an array of rna targets. squaritto et al found that a pool of ebp localized to the nucleolus binds rna and may be involved in ribosomal biogenesis. ebp can bind to b, as a component of pre- ribosomal ribonucleoprotein complexes. in the cytoplasm, ebp associates with the s subunit of mature ribosomes, suggesting that it can also be involved in protein translation. sedimentation studies revealed that ebp copurified with eif2α, a component of the translation initiation complex. ebp contains an rna binding domain σ- like motif that maps to aa 46– that mediates its interaction with rna and its nucleolar localization. a double-stranded rna binding domain was also mapped to aa 91– other studies showing that ebp binds the 3'utr of bcl- mrna and stabilizes β-globin-are bcl- transcripts suggest that ebp is a functional rna binding protein. finally, ebp has been demonstrated to bind to viral internal ribosome entry sites. despite burgeoning interest in the role of ebp in several important cellular activities, the physiological function of ebp in the intact organism is not known. we therefore characterized ebp1- deficient mice generated by a gene trap insertion in intron of the pa2g gene. we show that ebp1-deficient mice exhibit transient dwarfism. this is accompanied by a decrease in serum levels of igf- and igf binding proteins and changes in expression of several other genes associated with cell growth. these results provide in vivo evidence that ebp plays an important role in cellular growth.  disruption of the ebp gene ebp1+/- mice were generated at lexicon genetics from es cells containing a retroviral gene trap in the ebp locus as described previously. sequence analysis showed that the gene trap was inserted at a single site in the kb intron of the ebp gene. the insertion led to the expression of an ebp1-neomycin fusion molecule containing only the first amino acids that are encoded by exon of ebp that has no known biological function. the mice were genotyped by pcr with primers located at each side of the gene trap and in the ltr cassette. the absence of ebp mrna in ebp1-/- mice was confirmed by rt-pcr with β actin as an internal control on total mrna from day embryo fibroblasts. the transcript was present in fibroblasts from wild type, but not ebp1-/- mef. in addition, western blot analysis indicated that ebp protein was not expressed in knock out mefs. ebp expression in adult life examination of est data bases and our own published data indicate that ebp is ubiquitously expressed in normal adult mouse tissue. we therefore measured ebp protein expression in several organs to determine the penetrance of the knock out. we found as expected that ebp was expressed in adult lung and kidney of week old wild type mice. ebp expression was absent in the ebp1-/- mouse. breeding data and post natal lethality breeding data were obtained from crosses between both heterozygous and homozygous mice, since both ebp1+/- and ebp1-/-male and females were fertile. in all cases, the sex ratio was approximately 1: the genotypes of the pups from heterozygous pairs were present at approximately the expected ratio of 25:50:. survival was approximately equal for all three groups eight weeks after birth. male and female knock-out mice were fertile and inbred mating resulted in viable offspring. however, litter size was smaller for the ebp1-/- mice. litter size of wild type mice was ±. and for knock out mice, ±. in general, life expectancy, of knock out mice was indistinguishable compared with their littermates. ten mice of each genotype were maintained for one year. 9/ knock out and 10/ heterozygous and 10/ wild type mice survived. reduced growth of ebp1-/- mice the most striking feature of adult ebp1-/- mice is their initial smaller body size and in about 1/ of the mice the presence of kinky tails. the progeny of crosses between heterozygous parents were kept with the heterozygous mother and weighed sequentially. at day there was a significant reduction of approximately 30% in body weight for both males and females. mice were weaned at day after day mice began to catch up in size with wild type litter mates and at day there were no differences in weight. to rule out the possibility that competition during milk sucking was responsible for growth retardation, ebp1-/- males and females or wild type males and females were crossed. ko or wt progeny were housed alone with their mother. males and females were weighed and data pooled due to difficulties in determining sex at early time points. weights were the same for wt and ko mice at day however, at days and these mice also displayed a 25% reduction in weight. macro and microscopic appearance of organs to identify development defects and or pathological features, we examined the macro and microscopic appearance of a series of organs from adult ebp mice. there were no gross morphological differences between the ebp1+/+ and ebp1-/- mice at weeks of age. the examination of hematoxylin and eosin stained sections from organs revealed no histological changes in gut, kidney, lymph nodes, testes, liver, spleen, pancreas, skeletal muscle, lung, testis and ovaries. peripheral blood counts and blood chemistries were within normal limits. glucose levels were ± mg/dl for knock-out mice and ± mg/dl for wild type mice. proliferative behavior of mefs as ebp1-/- mice were small and the ebp gene was originally identified as a proliferation associated gene, we examined the proliferative behavior of day mouse embryo fibroblasts. under the standard culture conditions used, the number of mefs from ebp1-/- mice was lower at all the time points tested. in addition, we were able to propagate wild type mefs for passages, but ebp1-/- mefs for only a senescent morphology was observed at passage for knock out mefs. target gene and global expression profiling in mefs to obtain additional information about the cellular pathways involved in the phenotype of the mefs, we performed global expression profiling of matched day mefs. the expression data have been deposited with the geo data base as gse a total of genes were changed more than two fold. among the transcripts involved in growth regulation, we found that several members of the insulin, egfr, cyclin and mapk families were significantly changed. to verify these findings, we first examined the expression of cyclin d which has previously been shown to be repressed by ectopic expression of ebp in human breast cancer cell lines. we found by rt-pcr and quantitative pcr analysis that cyclin d mrna was increased. however, cyclin d protein levels were equal in wild type and knock out mefs reflecting the complex regulation of cyclin d protein expression. three regulators of the insulin signaling pathway were also found to be significantly decreased. these include igf- igfbp- and igfbp- the levels of igf- were measured by elisa in conditioned media derived from wild type and knock out mefs. the concentration of igf- in conditioned media from day cultures of mefs was ± pg/ml for knock out cells and ± pg/ml for wild type mefs. alterations of egf-regulated signaling pathways our microarray data indicated that two members of the mapk signal transduction pathway, sos and grb were upregulated in knock-out as compared with wild type mefs. we verified by western blot analysis that sos was increased in ebp knock out mefs as compared with wild type cells. we therefore tested if the cellular response to egf would be changed in ebp knock out mefs. murine mefs are known to express egfr and egfr mrna expression was not changed in ebp knock outs. cells were treated with egf for the times indicated and mapk activation assessed by detection of phosphorylated mapk by western blot analysis. the results indicated that both wild type and knock out mefs could respond to egf stimulation as evidenced by phosphorylation of mapk. however, mapk was basally phosphorylated only in knock out mefs. egf-induced akt phosphorylation in wild type and knock out cells was also determined using a phospho specific akt antibody. the response to egf was equivalent in wild type and knock out cells. changes in expression of proliferation associated genes in adult target tissues previous in vitro work with ebp has used breast and prostate cancer cell lines derived from adults. in these tissues, the expression of cyclin d, agr and androgen receptor have been shown to be regulated by ebp we therefore examined expression of these genes in adult mammary and prostate epithelium. we found that in mammary epithelial cells, cyclin d protein levels were not changed in knock out mice as compared to wild type. however, the phosphorylation of akt was greatly increased in ebp1-/- mammary tissue. in prostate cancer cell lines, the expression of the androgen receptor and the metastasis associated protein agr has been demonstrated to be decreased by ectopic expression of ebp we found that agr mrna and protein expression and ar protein expression were increased in prostates of ebp knock out mice as compared to age matched controls. finally, based on data from mefs, we examined serum levels of igf- from age matched wild type and knock out mice by elisa assays. igf- was reduced to 66% of wild type levels in serum of month old mice. in addition, western blot analysis of adult liver and kidney indicated that igfbp- was reduced in knock-out versus wild type organs. discussion to characterize the physiological role of ebp we generated ebp deficient mice exhibiting a gene trap introduced into intron of the ebp gene. the gene trap leads to deletion of all but the first amino acids of ebp which have no biological function on their own. the most striking feature of the ebp1-/- phenotype was the small size of the mice. it is of interest to note that inhibition of expression of the potato homologue of ebp also results in a dwarf phenotype. ebp1-/- pups were on average 25% smaller than their wild type and heterozygous littermates until day the approximate onset of puberty. mice were well proportioned with no skeletal abnormalities other than a kinky tail in approximately 1/ of the mice. an unusual feature of our in vivo analysis is the fact that the reduced weight was recovered after the prepubescent stage, while ebp is still normally transcribed and translated and therefore likely to have a regulatory role. although we are unable to explain the mechanism underlying this recovery, we report this observation as a possible example of temporal restricted competence in growth regulation of specific cell types by ebp this phenotype is similar to that observed for the otx gene, a homeobox containing gene that in vitro activates transcription of gh, fsh, and lh. it is of interest that the phenotype of ebp knock out animals also resembles that of the imp knock out mice. imp belongs to a family of rna binding proteins implicated in mrna localization, turnover, and translational control. these mice also exhibit a dwarf phenotype with reduced proliferation of knock out mefs. in these mice, growth retardation is related to a hypoplastic small intestine. in contrast, ebp mice exhibit a transient dwarfism and their intestines are both grossly and histologically normal at weeks. however, we were unable to assess intestinal anatomy and histology at earlier time points, and it remains possible that abnormalities occur when the weight differences are most marked. ebp has also been shown to be an rna binding protein with a sigma- like motif. this motif has been identified as a new rna binding domain unique to the superfamily of imp or brix proteins that are required for ribosome biogenesis. a role for ebp in ribosomal biogenesis is supported by studies indicating its localization in the nucleolus, its association with s ribosomal rna, and its complexing with proteins such as b which are involved in ribosome processing. whereas the significance of rna binding proteins in spatial and temporal control of gene expression during development is evident in invertebrate organisms, the function of rna binding proteins during mammalian development is less clear. with the exception of fmrp, dazla, and nova- which are associated with mental retardation, azospermia, and neuronal viability respectively, the majority of the mammalian rna binding proteins are constitutively expressed and exhibit a relative broad rna-binding specificity. further work is needed to uncover the role that ebp1's rna binding activity has in the development of this phenotype and the target rnas that specifically interact with ebp the molecular pathways leading to dwarfism are likely to involve several mechanisms. as reduced growth was manifested in the mefs from day embryos, we used these cells to measure changes in gene expression associated with loss of ebp global gene expression analysis indicated that expression of igf- and the igf binding proteins igfbp and was decreased. the in vivo growth promoting role of the igfs has been demonstrated conclusively from the dwarfing phenotypes observed after targeted mutagenesis of the mouse igf and igf1r genes.. igf- functions both during embryonic and post natal development. igf- mutants exhibit a birth weight that is 60% of normal and the difference between their size and that of wild type mice increases progressively with age. the ebp phenotype is not as severe as that observed in igf- deficient mice. the function of igfbps in cell growth is more complex. however, one role is to sequester and stabilize igf-. thus, decreases in igfbps may result in destabilization of igf- and may be partially responsible for lower levels of serum igf- we found that mapk was constitutively phosphorylated in mefs derived from knock out mice. this constitutive phosphorylation was associated with the overexpression of sos- an upstream activator of the ras -mapk signal transduction pathway. it is possible that overexpression of sos- led to increased basal phosphorylation of mapk. in contrast, both the basal and hrg-induced activation of the mapk pathway is increased in ebp transfected breast cancer cell lines. the reasons for these apparently discordant findings may be related to the ligand used, the differential expression of erbb receptors in breast cancer cells versus fibroblasts, and the cellular background. we have previously shown that inhibition of ebp expression results in increases in phospho akt in human breast cancer cell lines, whereas ectopic expression of ebp inhibits hrg induced akt activation. here, we found that ebp had no effect on akt phosphorylation after egf stimulation of mefs. however, akt was constitutively phosphorylated in mammary epithelial cells of knock-out mice. the mechanism of the increase in akt phosphorylation is unclear. paradoxically, ahn et al have shown the binding of ebp to nuclear akt increases akt phosphorylation in pc- neuronal cells. it is not known if ebp binds akt in normal mammary epithelial cells or affects its phosphorylation. these studies point up the importance of tissue context in the activation of specific signaling molecules. as activation of akt is important in development of mammary adenocarcinoma, we are currently monitoring breast tumor incidence with age in ebp knock out mice. we have not yet seen increased tumor incidence in a small cohort of mice that have been carried until one year. finally, our laboratory has demonstrated that ectopic expression of ebp results in decreased expression of ar and ar target genes and inhibition of growth of prostate cancer cells both in vivo and in vitro. in the current study, deletion of the ebp gene led to increased expression of ar in prostates of adult mice and upregulation of the metastasis associated gene agr ar plays a key role in the development and progression of prostate cancer, and we anticipate that the ebp knockout mouse may have a hyperactivated ar signaling axis that may result in increased incidence of prostate cancer. also with regard to prostate cancer, decreased expression of igfbp- is correlated with increased risk of developing prostate cancer. western blot analysis verified that ebp knockout led to reduced protein levels of igfbp- in liver and kidney. we were unable to detect igfbp- in prostates of either wild type or knock out mice, but studies are ongoing to assess if there are changes in igfbp- expression in the prostates of knock out mice. we are also monitoring the incidence of prostate cancer of the ebp1-/- mice as they age. nevertheless, current studies, together with our previous reports, strongly suggest that ebp as an endogenous regulator of erbb-ar crosstalk, may regulate expression of a series of genes that are involved in aggressive prostate cancer growth.  in summary, although ebp appears to be dispensable for prenatal development, loss of ebp affects post-natal growth. this growth delay may be related to changes in igf- and igfbp levels and to changes in key components of cellular proliferation pathways. of interest, two genes involved in prostate cancer progression, androgen receptor and agr are increased in prostates of ebp knock out mice. our ebp1-/- mouse line represents a new in vivo model to investigate ebp function in the entire organism. 
ebp1-/- mice were on average 30% smaller than wild type and heterozygous sex matched littermates. the ebp1-/- mouse line represents a new in vivo model to investigate ebp function in the whole organism. the erbb binding protein- belongs to a family of dna/rna binding proteins implicated in cell growth, apoptosis and differentiation. microarray expression analysis revealed changes in genes important in cell growth including members of the mapk signal transduction pathway. therefore, we generated ebp1-deficient mice carrying a gene trap insertion in intron of the ebp gene. these results indicate that ebp can affect growth in an animal model, but that the expression of proliferation related genes is cell and context specific. growth retardation was apparent from day until day igf- production and igbp- and protein levels were reduced in both embryo fibroblasts and adult knock-out mice. the proliferation of fibroblasts derived from day knock out embryos was also decreased as compared to that of wild type cells. however, the physiological role of ebp in the whole organism is not known. in addition, the expression or activation of proliferation related genes such as akt and the androgen receptor, previously demonstrated to be affected by ebp expression in vitro, was altered in adult tissues.
 the erbb binding protein- belongs to a family of dna/rna binding proteins implicated in cell growth, apoptosis and differentiation. however, the physiological role of ebp in the whole organism is not known. therefore, we generated ebp1-deficient mice carrying a gene trap insertion in intron of the ebp gene.  ebp1-/- mice were on average 30% smaller than wild type and heterozygous sex matched littermates. growth retardation was apparent from day until day igf- production and igbp- and protein levels were reduced in both embryo fibroblasts and adult knock-out mice. the proliferation of fibroblasts derived from day knock out embryos was also decreased as compared to that of wild type cells. microarray expression analysis revealed changes in genes important in cell growth including members of the mapk signal transduction pathway. in addition, the expression or activation of proliferation related genes such as akt and the androgen receptor, previously demonstrated to be affected by ebp expression in vitro, was altered in adult tissues.  these results indicate that ebp can affect growth in an animal model, but that the expression of proliferation related genes is cell and context specific. the ebp1-/- mouse line represents a new in vivo model to investigate ebp function in the whole organism. 