BACKGROUND
wnt ligands are secreted glycoproteins that control a wide range of processes in the developing embryo and in adult tissues.
aberrant wnt signaling is increasingly being implicated in numerous diseases, including malignancies, alzheimers disease, retinal degenerations and abnormal development of the eye, limbs and skeleton  <cit> .
characterizing proteins that regulate the wnt pathway have revealed important insights into wnt-dependent processes and potential directions towards novel therapies  <cit> .
the best understood of the major wnt pathways is the canonical/β-catenin pathway.
in the absence of wnt ligands, β-catenin levels are suppressed by the apc-axin-gsk3β protein complex via phosphorylation and subsequent degradation by the proteosome  <cit> .
β-catenin is a transcriptional cofactor and is also an essential component of cell-cell adhesion complexes.
wnt ligands bind to the cell surface receptors frizzled and ldl receptor related proteins  <dig> and  <dig> , leading to disheveled activation and ultimately reducing β-catenin degradation.
stabilized β-catenin is transported into the nucleus where it binds to tcf/lef type transcription factors and initiates transcription of wnt target genes.
the dickkopf  family of proteins, dkk <dig>   <dig>   <dig> and  <dig> and soggy, are secreted regulators of wnt signaling  <cit> .
the five dkk proteins share 37–50% protein identity and contain two conserved cysteine-rich regions separated by a variable linker region  <cit> .
dkk <dig>  dkk <dig> and dkk <dig> inhibit wnt signaling by binding to lrp5/ <dig> and the transmembrane protein kremen which results in lrp5/ <dig> internalization and prevents wnt and frizzled from forming an active complex with lrp5/ <dig>  <cit> .
dkk <dig> can also activate the wnt pathway in certain situations, depending on the cell type, the presence of wnt ligands and levels of lrp5/ <dig>  <cit> .
unlike its related family members, characterizing dkk <dig> activity has been elusive.
dkk <dig> did not regulate wnt signaling in various activity assays, including wnt-dependent secondary axis induction in xenopus embryos and wnt1/fz <dig> signaling in cultured cells  <cit> .
dkk <dig> also did not physicallyinteract with lrp5/ <dig> or kremen  <cit> .
however, caricasole et al demonstrated that dkk <dig> was a weak inhibitor of wnt7a signaling in pc <dig> cells although co-expression of lrp <dig> or lrp <dig> was required to uncover this activity  <cit> .
dkk <dig> displayed wnt inhibitor activity in the osteocarcinoma saos- <dig> cell line, measured by decreased cytoplasmic levels of β-catenin  <cit> , but did not inhibit wnt reporter tcf/lef luciferase activity assays in a prostate cancer cell line  <cit> .
therefore, the relationship between dkk <dig> and wnt signaling is unclear despite its sequence similarity to the other dkk genes.
dkk <dig> is expressed during embryonic development in many organs, including neural epithelium, limb bud, bone and heart, particularly in regions of epithelial-mesenchyme transformation  <cit> .
dkk <dig> is also widely expressed in adult tissues, with the highest levels found in the heart and brain  <cit> .
dkk3-deficient mice develop normally, are fertile and have a mild phenotype that includes hyperactivity, increased immunological and hematological markers and a slight decrease in lung ventilation  <cit> .
the absence of severe phenotypes in dkk <dig> knock-out mice may be due to compensation from the dkk <dig> homolog soggy  <cit> .
alternatively, physiological stress or injury may be required for the appearance of a dkk3-dependent phenotype.
in this study, we investigated the activity of dkk <dig> in wnt signaling and cell death.
we demonstrated two novel functions for dkk <dig>  first, dkk <dig> is a cell-specific positive regulator of the canonical wnt signaling pathway in primary cell culture and cell lines.
second, dkk <dig> protected transfected cells from apoptotic stress.
we also characterized the distribution of dkk <dig> in the retina and found that dkk <dig> is expressed highly in müller glia and ganglion cells during retinal development and in adult retina.
müller glia are the principle supportive glia in the retina and are believed to protect photoreceptors during retinal injury by secreting growth factors  <cit> .
we previously demonstrated that dkk <dig> transcripts were increased in a mouse model of retinal degeneration, particularly during cone photoreceptor death  <cit> .
furthermore, wnt signaling is upregulated during retinal degeneration in müller glia and wnt activators protect primary photoreceptor cultures from apoptosis  <cit> .
together, these results identify for the first time that dkk <dig> is a secreted pro-survival signaling protein, and suggest that dkk <dig> may play a role in müller glia activity in the retina.
RESULTS
dkk <dig> is expressed and secreted from retinal müller glia
cellular differentiation in the mouse retina into the major neuronal and glial cell types occurs at stereotypic time-points during the neo-natal period.
we used immunohistochemistry to characterize the distribution of dkk <dig> during post-natal retinal development, especially during the time of müller glia differentiation.
dkk <dig> immunoreactivity is present diffusely at post-natal day   <dig> and becomes concentrated in the developing synaptic layers in the outer plexiform layer  and ganglion cell layer  by p <dig>  and then remains in these locations through the beginning of müller glia differentiation  .
as müller glia cells continue to differentiate during p <dig> to p <dig>  dkk <dig> becomes more concentrated in the inner nuclear layer , where nuclei of the interneurons horizontal cells, bipolar cells and amacrine cells are located, and in the narrow margin composed of the photoreceptor inner segments and the external limiting membrane.
localization to this inner segment region  suggests that dkk <dig> is expressed in or secreted from müller glia end-feet.
by p <dig>  dkk <dig> appears associated with radial fibers extending across the retina, which correspond to müller glia processes.
dkk <dig> remained localized in the inner nuclear layer and the ganglion cell layer in adult mouse retina , consistent with our previous study localizing dkk <dig> transcript distribution  <cit> .
dkk <dig> staining in the adult also remained prominent in the synaptic plexiform layers between the onl and inl, and the inl and gcl, and the photoreceptor inner segment  region.
double immunostaining using antibodies against dkk <dig> and the müller glia marker glutamine synthetase  indicate that dkk <dig> colocalizes with gs-positive müller glia radial fibers and cell bodies .
the immunostaining in figures  <dig> and  <dig> used different anti-dkk <dig> antibodies  and the equivalent staining patterns confirm the specificity of dkk <dig> detection.
the specificity of the antibody was confirmed by western blotting, which detected a single band at the predicted size .
müller glia span the entire thickness of the tissue, ensheathing and interacting with all retinal neurons.
müller glia are vital to the normal retinal function by regulating ion concentrations, signaling molecules and neurotransmitters, mediating water transport, and removing glutamate and scavenging free radicals.
during retinal degeneration müller glia are believed to secrete pro-survival growth factors that protect remaining photoreceptors from injury.
to confirm dkk <dig> expression in glia we cultured müller glia from rat and mouse retinas.
one hundred percent of the müller glia showed colocalization of dkk <dig> with the müller glia marker protein glutamine synthetase .
dkk <dig> was distributed along processes and also in a punctate peri-nuclear pattern, consistent with the endoplasmic reticulum/golgi complex.
this peri-nuclear localization is evident in a higher magnification image of the human müller glia cell line mio-m <dig>  <cit>  .
western blotting confirmed that dkk <dig> is secreted  from mio-m <dig> cultures .
dkk <dig> regulates wnt signaling
wnt signaling was measured using a transcriptional reporter assay with the top-flash luciferase plasmid.
mio-m <dig> müller glia cells were cotransfected with top-flash and lacz plasmids along with dkk <dig>  dkk <dig> or a control gene .
at  <dig> hrs post-transfection, the transfected cells were incubated with wnt3a-containing conditioned media or control conditioned media that lacks the wnt3a ligand.
wnt signaling was induced by wnt3a conditioned media in the mio-m <dig> human müller glia cell line , indicating that these cells are wnt-responsive, as we recently demonstrated  <cit> .
the mutated control reporter fop-flash did not show a response to wnt3a, as expected .
dkk <dig> reduced wnt3a-induced signaling in mio-m <dig> cells by approximately five-fold  compared with gfp expression.
in contrast, dkk <dig> induced approximately two-fold higher wnt signaling compared with gfp .
interestingly, dkk <dig> potentiating activity was cell-type specific.
in contrast to the mio-m <dig> cells, dkk <dig> had a small inhibitory, although not significant, effect on wnt3a-induced signaling in the cos <dig> cell line and reduced luciferase activity by 33%compared with control tr ansfections .
dkk <dig> decreased luciferase activity by 67% in cos <dig> cells .
this result suggests that cos <dig> cells do not have the molecular mechanisms to convey dkk <dig> potentiating activity or that the inhibitor activity of dkk <dig> is greater than, and over-rides, its potentiating activity.
dkk <dig> increased wnt3a-induced signaling in primary müller glia isolated from mouse retina.
in these experiments we tested the activity of dkk <dig> when it was added to the glia cells as conditioned media.
hek <dig> cells stably expressing dkk <dig> were created and used as a source of dkk <dig>  <cit>  .
dkk <dig> treatment in the absence of wnt3a produced baseline levels of wnt signaling and was equivalent to control media not containing dkk <dig> .
dkk <dig> by itself had baseline levels in all the cell types tested, indicating that dkk <dig> potentiates the wnt pathway when it is stimulated by wnt3a but cannot initiate signaling on its own.
incubating primary müller glia in dkk3-containing conditioned media increased wnt signaling by  <dig> -fold ).
.
although co-stimulation with a wnt ligand was required to demonstrate dkk <dig> activity, over-expression of wnt receptors was not necessary, in contrast to dkk <dig> activity in pc <dig> cells  <cit> .
to begin to explore the mechanism of wnt regulation by dkk <dig>  we tested whether dkk <dig> blocks dkk <dig> activity.
for this experiment we used a dkk <dig> stable cell line made in hek <dig> cells to ensure that the majority of cells coexpressed dkk <dig> and dkk <dig>  we previously demonstrated that dkk <dig> potentiated wnt signaling in this cell type <cit> .
wnt3a ligand induced significantly more wnt signaling in the cells expressing dkk <dig> than the control cell line made with the empty vector .
if dkk <dig> acts independently from dkk <dig> then we expect that dkk <dig> would not fully inhibit wnt signaling in dkk3-expressing cells compared with the vector cell line, and that the combination of dkk <dig> + dkk <dig> + wnt3a would be less inhibited and have more wnt signaling than dkk <dig> + wnt3a in the vector cell line.
as shown in figure  <dig>  dkk <dig> inhibited wnt3a-induced signaling to an equivalent extent in the dkk <dig> and vector stable cell lines.
this result suggests that dkk3-mediated potentiation of wnt3a signaling is dependent on, or occurs upstream from, a dkk1-controlled pathway or receptor.
finally, it is unlikely that the transfection method influenced the effect of dkk <dig> because there is no correlation between dkk <dig> activity and lipofectamine or electroporation.
for example, dkk <dig> had opposite effects in hek <dig> and cos <dig> cells despite both being transfected with lipofectamine, whereas dkk <dig> activity was similar in mio-m <dig> cells as hek <dig> despite the former being transfected using electroporation.
dkk <dig> regulates cell death
we next investigated whether dkk <dig> plays a role in cell survival by testing the effect of dkk <dig> expression on cell death induced by h2o <dig> and staurosporine.
the hek <dig> cell line was used because it has dkk3-responsive wnt signaling , indicating the potential presence of dkk <dig> receptors and signaling proteins.
hek <dig> cells were transiently transfected with dkk <dig> or the control genes lacz or bclxl and were exposed to  <dig>  to  <dig>  mm h2o <dig> for  <dig> hr.
dkk <dig> expression significantly increased viability in cells exposed to h2o <dig>  compared with the lacz transfection control .
indeed, the pro-survival effect of dkk <dig> was equivalent to the anti-apoptotic gene bclxl used as the positive control.
the baseline viability of hek <dig> cells transfected with dkk <dig> and vector were equivalent in the absence of apoptotic inducers .
dkk <dig> transfection also decreased apoptosis induced by staurosporine, a well-characterized pro-apoptotic protein kinase inhibitor.
dkk <dig> expression reduced caspase activation by 4-fold in hek <dig> cells, compared with the vector control .
protection from apoptosis was also observed in three independent clones of hek <dig> cell lines stably expressing dkk <dig> .
however, dkk <dig> did not reduce caspase activity in cos <dig> cells .
therefore, dkk <dig> demonstrates cell-specific protection, similar to its cell-specific potentiation of wnt signaling.
discussion and
CONCLUSIONS
the function of dkk <dig> and its role in wnt signaling had been unclear despite it sharing approximately 40% protein identity to the well-characterized dkk <dig> and dkk <dig> wnt inhibitors.
in the present study, we found that dkk <dig> is a cell-type specific regulator of wnt signaling.
dkk <dig> increased wnt activation in primary müller glia cultures, a müller glia cell line and in hek <dig> cells, but not in cos <dig> cells.
dkk <dig> activity was not dependent on the method of delivery because transient and stable transfections of dkk <dig> plasmids and treatment with dkk <dig> conditioned media demonstrated equivalent results.
although the effect of dkk <dig> on wnt signaling may be indirect, to our knowledge this is the first demonstration that dkk <dig> acts as a cell-specific positive regulator of the wnt pathway.
dkk <dig> was originally proposed to be a divergent, non-wnt regulating dkk family member  <cit>  although later studies indicated that dkk <dig> inhibits wnt signaling in pc <dig> and osteocarcinoma saos- <dig> cells  <cit> .
discrepant effects on the wnt pathway have been observed for other wnt regulator genes.
for example, dkk <dig> activates wnt signaling when coexpressed with certain frizzled and lrp <dig> receptors but inhibits wnt signaling in other situations  <cit> .
wnt <dig> also activates canonical wnt signaling in some cell lines but inhibits wnt signaling in others  <cit> .
furthermore, the non-canonical wnt/ca2+ pathway activator wnt5a activates canonical wnt signaling in hek <dig> cells when the co-receptors fz <dig> and lrp <dig> are over-expressed, but inhibits canonical signaling in the absence of these receptors  <cit> .
therefore, wnt signaling activation in vitro, and potentially also in vivo, is not a sole property of the ligand or regulator but appears to be related to other factors, including receptor availability  <cit> .
receptor context influences the regulation of canonical wnt signaling by dkk <dig>  <cit>  and wnt5a  <cit> , suggesting that specific receptor components in hek <dig> and müller glia, such as a putative dkk <dig> receptor, may mediate the wnt-inducing activity of dkk <dig>  absence or lower expression of these components may cause its inhibitory activity in cos <dig>  and pc <dig> cells  <cit>  and its inactivity in other assays.
although dkk <dig> activity was blocked by dkk <dig> coexpression in hek <dig> cells, the mechanism of wnt modulation by dkk <dig> may differ from dkk <dig> and dkk <dig> because dkk <dig> does not bind to kremen or lrp proteins  <cit> .
future studies will investigate the factors that are required for dkk <dig> activity.
we demonstrated that dkk <dig> has pro-survival properties in hek <dig> cells but not in cos <dig> cells.
this cellular difference between dkk <dig> protection also suggests that cos <dig> cells lack the appropriate dkk <dig> receptors, although this cell type does express endogenous dkk <dig> .
although the wnt pathway itself is protective in most tissues  <cit> , dkk3-mediated cellular protection is unlikely to be dependent on wnt signaling because wnt3a was not added in the apoptosis assays.
however, we cannot exclude the possibility that low levels of wnt activators are present in the culture serum.
several reports demonstrated that dkk <dig> regulates cell death in cancer cells, protecting osteocarcinoma cells against serum starvation and chemotherapy agents  <cit>  but inducing apoptosis in liver cancer cell lines  <cit> .
in our study, dkk <dig> expression alone did not increase basal viability, suggesting that it plays a reactive role after apoptosis has begun.
interestingly, dkk3-deficient mice did not display any phenotypes that would indicate defects in cell survival or altered wnt signaling  <cit> .
furthermore, these mice had normal retinal development.
if dkk <dig> is protective only in response to retinal injury then tissue damage may be required to reveal a phenotype.
alternatively, dkk3-mediated protection may be limited to specific cell types in culture.
müller glia secrete pro-survival growth factors during retinal degeneration that are believed to rescue remaining photoreceptors in a toxic environment  <cit> .
we recently demonstrated that wnt signaling is upregulated during retinal degeneration in müller glia and wnt activators protect primary photoreceptor cultures from apoptosis  <cit> .
the localization of dkk <dig> to müller glia and its increased expression during retinal degeneration, combined with the functional properties identified above, raise the intriguing possibility that dkk <dig> may be a component of müller glia-mediated retinal protection.
such protection could occur directly by acting on vulnerable photoreceptors, or indirectly by activating wnt signaling.
the glia may secrete dkk <dig> in vivo which could then increase wnt signaling in fellow glia or in neighboring cell types.
although the cell lines are not an appropriate model for photoreceptors, we have achieved our goal of defining roles of dkk <dig> in cultured cells as a first approach towards determining the potential activity of dkk <dig> in the retina.
manipulating dkk <dig> expression in normal and degenerating retina will provide insight the role of dkk <dig> in the retina.

 wnt ligands are secreted glycoproteins that control a wide range of processes in the developing embryo and in adult tissues. aberrant wnt signaling is increasingly being implicated in numerous diseases, including malignancies, alzheimers disease, retinal degenerations and abnormal development of the eye, limbs and skeleton. characterizing proteins that regulate the wnt pathway have revealed important insights into wnt-dependent processes and potential directions towards novel therapies. the best understood of the major wnt pathways is the canonical/β-catenin pathway. in the absence of wnt ligands, β-catenin levels are suppressed by the apc-axin-gsk3β protein complex via phosphorylation and subsequent degradation by the proteosome. β-catenin is a transcriptional cofactor and is also an essential component of cell-cell adhesion complexes. wnt ligands bind to the cell surface receptors frizzled and ldl receptor related proteins and, leading to disheveled activation and ultimately reducing β-catenin degradation. stabilized β-catenin is transported into the nucleus where it binds to tcf/lef type transcription factors and initiates transcription of wnt target genes. the dickkopf family of proteins, dkk and and soggy, are secreted regulators of wnt signaling. the five dkk proteins share 37–50% protein identity and contain two conserved cysteine-rich regions separated by a variable linker region. dkk dkk and dkk inhibit wnt signaling by binding to lrp5/ and the transmembrane protein kremen which results in lrp5/ internalization and prevents wnt and frizzled from forming an active complex with lrp5/. dkk can also activate the wnt pathway in certain situations, depending on the cell type, the presence of wnt ligands and levels of lrp5/. unlike its related family members, characterizing dkk activity has been elusive. dkk did not regulate wnt signaling in various activity assays, including wnt-dependent secondary axis induction in xenopus embryos and wnt1/fz signaling in cultured cells. dkk also did not physicallyinteract with lrp5/ or kremen. however, caricasole et al demonstrated that dkk was a weak inhibitor of wnt7a signaling in pc cells although co-expression of lrp or lrp was required to uncover this activity. dkk displayed wnt inhibitor activity in the osteocarcinoma saos- cell line, measured by decreased cytoplasmic levels of β-catenin, but did not inhibit wnt reporter tcf/lef luciferase activity assays in a prostate cancer cell line. therefore, the relationship between dkk and wnt signaling is unclear despite its sequence similarity to the other dkk genes. dkk is expressed during embryonic development in many organs, including neural epithelium, limb bud, bone and heart, particularly in regions of epithelial-mesenchyme transformation. dkk is also widely expressed in adult tissues, with the highest levels found in the heart and brain. dkk3-deficient mice develop normally, are fertile and have a mild phenotype that includes hyperactivity, increased immunological and hematological markers and a slight decrease in lung ventilation. the absence of severe phenotypes in dkk knock-out mice may be due to compensation from the dkk homolog soggy. alternatively, physiological stress or injury may be required for the appearance of a dkk3-dependent phenotype. in this study, we investigated the activity of dkk in wnt signaling and cell death. we demonstrated two novel functions for dkk first, dkk is a cell-specific positive regulator of the canonical wnt signaling pathway in primary cell culture and cell lines. second, dkk protected transfected cells from apoptotic stress. we also characterized the distribution of dkk in the retina and found that dkk is expressed highly in müller glia and ganglion cells during retinal development and in adult retina. müller glia are the principle supportive glia in the retina and are believed to protect photoreceptors during retinal injury by secreting growth factors. we previously demonstrated that dkk transcripts were increased in a mouse model of retinal degeneration, particularly during cone photoreceptor death. furthermore, wnt signaling is upregulated during retinal degeneration in müller glia and wnt activators protect primary photoreceptor cultures from apoptosis. together, these results identify for the first time that dkk is a secreted pro-survival signaling protein, and suggest that dkk may play a role in müller glia activity in the retina.  dkk is expressed and secreted from retinal müller glia cellular differentiation in the mouse retina into the major neuronal and glial cell types occurs at stereotypic time-points during the neo-natal period. we used immunohistochemistry to characterize the distribution of dkk during post-natal retinal development, especially during the time of müller glia differentiation. dkk immunoreactivity is present diffusely at post-natal day and becomes concentrated in the developing synaptic layers in the outer plexiform layer and ganglion cell layer by p and then remains in these locations through the beginning of müller glia differentiation. as müller glia cells continue to differentiate during p to p dkk becomes more concentrated in the inner nuclear layer, where nuclei of the interneurons horizontal cells, bipolar cells and amacrine cells are located, and in the narrow margin composed of the photoreceptor inner segments and the external limiting membrane. localization to this inner segment region suggests that dkk is expressed in or secreted from müller glia end-feet. by p dkk appears associated with radial fibers extending across the retina, which correspond to müller glia processes. dkk remained localized in the inner nuclear layer and the ganglion cell layer in adult mouse retina, consistent with our previous study localizing dkk transcript distribution. dkk staining in the adult also remained prominent in the synaptic plexiform layers between the onl and inl, and the inl and gcl, and the photoreceptor inner segment region. double immunostaining using antibodies against dkk and the müller glia marker glutamine synthetase indicate that dkk colocalizes with gs-positive müller glia radial fibers and cell bodies. the immunostaining in figures and used different anti-dkk antibodies and the equivalent staining patterns confirm the specificity of dkk detection. the specificity of the antibody was confirmed by western blotting, which detected a single band at the predicted size. müller glia span the entire thickness of the tissue, ensheathing and interacting with all retinal neurons. müller glia are vital to the normal retinal function by regulating ion concentrations, signaling molecules and neurotransmitters, mediating water transport, and removing glutamate and scavenging free radicals. during retinal degeneration müller glia are believed to secrete pro-survival growth factors that protect remaining photoreceptors from injury. to confirm dkk expression in glia we cultured müller glia from rat and mouse retinas. one hundred percent of the müller glia showed colocalization of dkk with the müller glia marker protein glutamine synthetase. dkk was distributed along processes and also in a punctate peri-nuclear pattern, consistent with the endoplasmic reticulum/golgi complex. this peri-nuclear localization is evident in a higher magnification image of the human müller glia cell line mio-m. western blotting confirmed that dkk is secreted from mio-m cultures. dkk regulates wnt signaling wnt signaling was measured using a transcriptional reporter assay with the top-flash luciferase plasmid. mio-m müller glia cells were cotransfected with top-flash and lacz plasmids along with dkk dkk or a control gene. at hrs post-transfection, the transfected cells were incubated with wnt3a-containing conditioned media or control conditioned media that lacks the wnt3a ligand. wnt signaling was induced by wnt3a conditioned media in the mio-m human müller glia cell line, indicating that these cells are wnt-responsive, as we recently demonstrated. the mutated control reporter fop-flash did not show a response to wnt3a, as expected. dkk reduced wnt3a-induced signaling in mio-m cells by approximately five-fold compared with gfp expression. in contrast, dkk induced approximately two-fold higher wnt signaling compared with gfp. interestingly, dkk potentiating activity was cell-type specific. in contrast to the mio-m cells, dkk had a small inhibitory, although not significant, effect on wnt3a-induced signaling in the cos cell line and reduced luciferase activity by 33%compared with control tr ansfections. dkk decreased luciferase activity by 67% in cos cells. this result suggests that cos cells do not have the molecular mechanisms to convey dkk potentiating activity or that the inhibitor activity of dkk is greater than, and over-rides, its potentiating activity. dkk increased wnt3a-induced signaling in primary müller glia isolated from mouse retina. in these experiments we tested the activity of dkk when it was added to the glia cells as conditioned media. hek cells stably expressing dkk were created and used as a source of dkk. dkk treatment in the absence of wnt3a produced baseline levels of wnt signaling and was equivalent to control media not containing dkk. dkk by itself had baseline levels in all the cell types tested, indicating that dkk potentiates the wnt pathway when it is stimulated by wnt3a but cannot initiate signaling on its own. incubating primary müller glia in dkk3-containing conditioned media increased wnt signaling by -fold ).. although co-stimulation with a wnt ligand was required to demonstrate dkk activity, over-expression of wnt receptors was not necessary, in contrast to dkk activity in pc cells. to begin to explore the mechanism of wnt regulation by dkk we tested whether dkk blocks dkk activity. for this experiment we used a dkk stable cell line made in hek cells to ensure that the majority of cells coexpressed dkk and dkk we previously demonstrated that dkk potentiated wnt signaling in this cell type. wnt3a ligand induced significantly more wnt signaling in the cells expressing dkk than the control cell line made with the empty vector. if dkk acts independently from dkk then we expect that dkk would not fully inhibit wnt signaling in dkk3-expressing cells compared with the vector cell line, and that the combination of dkk + dkk + wnt3a would be less inhibited and have more wnt signaling than dkk + wnt3a in the vector cell line. as shown in figure dkk inhibited wnt3a-induced signaling to an equivalent extent in the dkk and vector stable cell lines. this result suggests that dkk3-mediated potentiation of wnt3a signaling is dependent on, or occurs upstream from, a dkk1-controlled pathway or receptor. finally, it is unlikely that the transfection method influenced the effect of dkk because there is no correlation between dkk activity and lipofectamine or electroporation. for example, dkk had opposite effects in hek and cos cells despite both being transfected with lipofectamine, whereas dkk activity was similar in mio-m cells as hek despite the former being transfected using electroporation. dkk regulates cell death we next investigated whether dkk plays a role in cell survival by testing the effect of dkk expression on cell death induced by h2o and staurosporine. the hek cell line was used because it has dkk3-responsive wnt signaling, indicating the potential presence of dkk receptors and signaling proteins. hek cells were transiently transfected with dkk or the control genes lacz or bclxl and were exposed to to mm h2o for hr. dkk expression significantly increased viability in cells exposed to h2o compared with the lacz transfection control. indeed, the pro-survival effect of dkk was equivalent to the anti-apoptotic gene bclxl used as the positive control. the baseline viability of hek cells transfected with dkk and vector were equivalent in the absence of apoptotic inducers. dkk transfection also decreased apoptosis induced by staurosporine, a well-characterized pro-apoptotic protein kinase inhibitor. dkk expression reduced caspase activation by 4-fold in hek cells, compared with the vector control. protection from apoptosis was also observed in three independent clones of hek cell lines stably expressing dkk. however, dkk did not reduce caspase activity in cos cells. therefore, dkk demonstrates cell-specific protection, similar to its cell-specific potentiation of wnt signaling. discussion and  the function of dkk and its role in wnt signaling had been unclear despite it sharing approximately 40% protein identity to the well-characterized dkk and dkk wnt inhibitors. in the present study, we found that dkk is a cell-type specific regulator of wnt signaling. dkk increased wnt activation in primary müller glia cultures, a müller glia cell line and in hek cells, but not in cos cells. dkk activity was not dependent on the method of delivery because transient and stable transfections of dkk plasmids and treatment with dkk conditioned media demonstrated equivalent results. although the effect of dkk on wnt signaling may be indirect, to our knowledge this is the first demonstration that dkk acts as a cell-specific positive regulator of the wnt pathway. dkk was originally proposed to be a divergent, non-wnt regulating dkk family member although later studies indicated that dkk inhibits wnt signaling in pc and osteocarcinoma saos- cells. discrepant effects on the wnt pathway have been observed for other wnt regulator genes. for example, dkk activates wnt signaling when coexpressed with certain frizzled and lrp receptors but inhibits wnt signaling in other situations. wnt also activates canonical wnt signaling in some cell lines but inhibits wnt signaling in others. furthermore, the non-canonical wnt/ca2+ pathway activator wnt5a activates canonical wnt signaling in hek cells when the co-receptors fz and lrp are over-expressed, but inhibits canonical signaling in the absence of these receptors. therefore, wnt signaling activation in vitro, and potentially also in vivo, is not a sole property of the ligand or regulator but appears to be related to other factors, including receptor availability. receptor context influences the regulation of canonical wnt signaling by dkk and wnt5a, suggesting that specific receptor components in hek and müller glia, such as a putative dkk receptor, may mediate the wnt-inducing activity of dkk absence or lower expression of these components may cause its inhibitory activity in cos and pc cells and its inactivity in other assays. although dkk activity was blocked by dkk coexpression in hek cells, the mechanism of wnt modulation by dkk may differ from dkk and dkk because dkk does not bind to kremen or lrp proteins. future studies will investigate the factors that are required for dkk activity. we demonstrated that dkk has pro-survival properties in hek cells but not in cos cells. this cellular difference between dkk protection also suggests that cos cells lack the appropriate dkk receptors, although this cell type does express endogenous dkk. although the wnt pathway itself is protective in most tissues, dkk3-mediated cellular protection is unlikely to be dependent on wnt signaling because wnt3a was not added in the apoptosis assays. however, we cannot exclude the possibility that low levels of wnt activators are present in the culture serum. several reports demonstrated that dkk regulates cell death in cancer cells, protecting osteocarcinoma cells against serum starvation and chemotherapy agents but inducing apoptosis in liver cancer cell lines. in our study, dkk expression alone did not increase basal viability, suggesting that it plays a reactive role after apoptosis has begun. interestingly, dkk3-deficient mice did not display any phenotypes that would indicate defects in cell survival or altered wnt signaling. furthermore, these mice had normal retinal development. if dkk is protective only in response to retinal injury then tissue damage may be required to reveal a phenotype. alternatively, dkk3-mediated protection may be limited to specific cell types in culture. müller glia secrete pro-survival growth factors during retinal degeneration that are believed to rescue remaining photoreceptors in a toxic environment. we recently demonstrated that wnt signaling is upregulated during retinal degeneration in müller glia and wnt activators protect primary photoreceptor cultures from apoptosis. the localization of dkk to müller glia and its increased expression during retinal degeneration, combined with the functional properties identified above, raise the intriguing possibility that dkk may be a component of müller glia-mediated retinal protection. such protection could occur directly by acting on vulnerable photoreceptors, or indirectly by activating wnt signaling. the glia may secrete dkk in vivo which could then increase wnt signaling in fellow glia or in neighboring cell types. although the cell lines are not an appropriate model for photoreceptors, we have achieved our goal of defining roles of dkk in cultured cells as a first approach towards determining the potential activity of dkk in the retina. manipulating dkk expression in normal and degenerating retina will provide insight the role of dkk in the retina. 
this unique dkk activity was blocked by co-expression of dkk additionally, dkk displayed pro-survival properties by decreasing caspase activation and increasing viability in hek cells exposed to staurosporine and h2o in contrast, dkk did not protect cos cells from apoptosis. we demonstrated that dkk potentiated wnt signaling in müller glia and hek cells but not in cos cells, indicating that it is a cell-type specific regulator of wnt signaling. we recently demonstrated that dkk transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. in this study, we characterized the activity of dkk in wnt signaling and cell death. although the biological properties of dickkopf and dickkopf are well characterized, little is known about the function of the related dickkopf protein in vivo or in cell lines. dkk was localized to müller glia and retinal ganglion cells in developing and adult mouse retina. western blotting confirmed that dkk is secreted from müller glia cells in culture. these data demonstrate that dkk is a positive regulator of wnt signaling, in contrast to its family member dkk furthermore, dkk protects against apoptosis by reducing caspase activity, suggesting that dkk may play a cytoprotective role in the retina.
 the wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. a major class of wnt signaling regulators is the dickkopf family of secreted glycoproteins. although the biological properties of dickkopf and dickkopf are well characterized, little is known about the function of the related dickkopf protein in vivo or in cell lines. we recently demonstrated that dkk transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. in this study, we characterized the activity of dkk in wnt signaling and cell death.  dkk was localized to müller glia and retinal ganglion cells in developing and adult mouse retina. western blotting confirmed that dkk is secreted from müller glia cells in culture. we demonstrated that dkk potentiated wnt signaling in müller glia and hek cells but not in cos cells, indicating that it is a cell-type specific regulator of wnt signaling. this unique dkk activity was blocked by co-expression of dkk additionally, dkk displayed pro-survival properties by decreasing caspase activation and increasing viability in hek cells exposed to staurosporine and h2o in contrast, dkk did not protect cos cells from apoptosis.  these data demonstrate that dkk is a positive regulator of wnt signaling, in contrast to its family member dkk furthermore, dkk protects against apoptosis by reducing caspase activity, suggesting that dkk may play a cytoprotective role in the retina. 