BACKGROUND
podocytes, also called visceral glomerular epithelial cells, are terminally differentiated cells overlaying the outer region of the glomerular basement membrane of renal glomeruli.
these cells have several key functions including the prevention of proteinuria, synthesis of basement membrane components, regulation of glomerular filtration, and counteraction of intraglomerular hydrostatic pressure  <cit> .
podocyte injury is typically associated with proteinuria and progressive glomerulosclerosis  <cit> .
podocytes are derived from epithelial cells originating in the metanephric mesenchyme, which develop into postmitotic terminally differentiated cells, and therefore have similarities to neurons  <cit> .
during glomerulogenesis, podocytes proliferate until the s-shape body stage and exit the cell cycle at the capillary loop stage  <cit> .
podocytes then acquire their fully differentiated phenotype, a process that is not complete until 1 week after birth in the mouse.
mature podocytes tightly regulate and maintain their quiescent and differentiated phenotype, and therefore lost podocytes cannot be replaced by proliferation of neighboring undamaged cells.
indeed, studies have shown that the inability to proliferate contributes to glomerular scarring  <cit> .
thus, the mechanism responsible for the cell cycle arrest that occurs during nephrogenesis may also participate in maintenance of cell cycle quiescence in mature podocytes.
megakaryoblastic leukemia  <dig>  was originally found in a study of a chromosomal translocation, t , which is closely related to the incidence of acute megakaryoblastic leukemia in infants and children  <cit> .
mkl <dig> has been recently shown to be a member of a three-protein family that includes mkl <dig> and myocardin.
these myocardin/mkl proteins serve as serum response factor  coactivators by binding to srf and strongly activating srf target genes  <cit> .
in contrast to myocardin, which has cardiac and smooth muscle-specific expression  <cit> , mkl <dig> and mkl <dig> are expressed in a wide range of embryonic and adult tissues  <cit> .
mkl <dig> regulates many processes including muscle cell differentiation  <cit> , cardiovascular development  <cit> , remodeling of neuronal networks in the developing and adult brain  <cit> , megakaryocytic differentiation and migration  <cit> , modulation of cellular motile functions, and epithelial-mesenchymal transition  <cit> .
notably, there is increasing evidence of the involvement of the myocardin/mkl family in suppression of cell proliferation and cell cycle progression.
both myocardin and mkl <dig> exert anti-proliferative effects in various cell lines  <cit> .
therefore, unraveling the functional pathways in which these proteins have a role and furthering our comprehension of the cellular mechanisms intrinsic to their regulation of cell proliferation will become increasingly important.
in the present study, we found that mkl <dig> expression was upregulated during temperature-switched growth arrest in murine podocyte clone  <dig>  cells.
overexpression of mkl <dig> resulted in inhibition of g1/s cell cycle progression in cell viability and edu cell proliferation assays, whereas mkl <dig> knockdown had the opposite effect.
we further demonstrated that mkl <dig> induced expression of the cyclin-dependent kinase inhibitor  p <dig> during the regulation of cell cycle arrest.
importantly, mkl <dig> expression was observed in podocytes of the mouse kidney during postnatal development, which was upregulated during the morphological switch of podocytes from proliferation to differentiation.
RESULTS
expression of mkl <dig> is upregulated during temperature-switched cell growth arrest in mpc <dig> cells
to assess the possible role of myocardin/mkl proteins in podocyte growth arrest, mpc <dig> cells were respectively maintained at the permissive temperature of 33°c and at the nonpermissive temperature of 37°c for 10 days.
as shown in figure 1a, a cell viability assay indicated that mpc <dig> cells cultured at 37°c showed a significant decrease in cell number compared with those cultured at 33°c.
the results of immunofluorescence staining in the edu cell proliferation assay further revealed a marked decrease in the number of cells in s phase by the temperature switch to 37°c .
the percentage of cells in s phase decreased from  <dig> % to  <dig> % as early as 2 days after the temperature switch.
at 4– <dig> days, the percentage of cells in s phase further decreased from  <dig> % to  <dig> %, which is consistent with a previous report indicating that the temperature switch induces growth arrest of podocytes in vitro  <cit> .figure  <dig>
mkl <dig> is upregulated during temperature-switched cell cycle arrest in mpc <dig> cells.
a) mpc <dig> cells were cultured at the permissive temperature of 33°c or the nonpermissive temperature of 37°c.
at the indicated time points, cell growth was measured using a cck- <dig> assay.
**p <  <dig>  compared with the control .
b) mpc <dig> cells were shifted from 33°c to 37°c and cultured for the indicated times.
cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars, 25 μm.
c) the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
**p <  <dig>  compared with the control .
d) mpc <dig> cells were shifted from 33°c to 37°c and cultured for the indicated times.
the mrna expression levels of myocardin, mkl <dig> and mkl <dig> were verified by qpcr.
gapdh was used to normalize expression levels.
*p <  <dig>  compared with the control .
e) mpc <dig> cells were shifted from 33°c to 37°c and cultured for the indicated times.
protein expression levels of mkl <dig> were examined by western blotting.
actin was used to normalize mkl <dig> levels.
the expression of myocardin/mkl proteins was then measured during temperature-switched growth arrest in mpc <dig> cells.
qpcr analysis indicated that the temperature switch to 37°c induced an approximate  <dig> -fold increase in mkl <dig> mrna expression compared with the basal level at 2 days .
at 4– <dig> days, mkl <dig> expression showed a 2-4-fold increase at the mrna level.
western blotting was used to confirm the upregulation of mkl <dig> expression at the protein level .
however, the alteration in myocardin and mkl <dig> expression was not as evident .
considering the dominant presence of mkl <dig> over its other family members, we focused on the effects of mkl <dig> in subsequent experiments.
mkl <dig> functions as an effective inducer of cell growth arrest in mpc <dig> cells
next, a mouse mkl <dig> expression plasmid  <cit>  was transiently transfected into mpc <dig> cells.
overexpression of mkl <dig> was assessed by western blotting .
compared with control cells, the cell viability assay indicated that ectopic expression of mkl <dig> inhibited mpc <dig> cell proliferation .
results of dna analysis by flow cytometry further confirmed that mkl1-overexpressing mpc <dig> cells had a lower population of s phase cells and a higher population of g0/g <dig> phase cells .
the edu cell proliferation assay revealed a marked decrease in the number of s phase cells after mkl <dig> overexpression .
the percentage of cells in s phase decreased from  <dig> % to  <dig> % at 72 h after transfection of the mkl <dig> expression plasmid.
furthermore, mpc <dig> cells were stably transfected with either the mkl <dig> expression plasmid  or the empty vector .
overexpression of mkl <dig> was then examined by western blotting .
cell viability and edu cell proliferation assays confirmed that mkl <dig> overexpression induced a delay in g1/s phase transition of mpc <dig> cells .figure  <dig>
overexpression of mkl <dig> induces mpc <dig> cell growth arrest.
a) mpc <dig> cells were transiently transfected with a mouse mkl <dig> expression plasmid and cultured at 33°c.
expression of mkl <dig> protein was verified by western blotting.
actin was used to normalize mkl <dig> levels.
b) at the indicated time points, cell growth was measured using the cck- <dig> assay.
*p <  <dig>  and **p <  <dig>  compared with the control .
c) cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars, 25 μm.
the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
*p <  <dig>  compared with the control .
d) mpc <dig> cells were stably transfected with a mouse mkl <dig> expression plasmid and cultured at 33°c.
expression of mkl <dig> protein was verified by western blotting.
actin was used to normalize mkl <dig> levels.
e) at the indicated time points, cell growth was measured using the cck- <dig> assay.
**p <  <dig>  compared with the control .
f) cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars, 25 μm.
the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
*p <  <dig>  compared with the control .
therefore, we hypothesized that knockdown of mkl <dig> by rna interference would result in an increase in the number of cells in s phase.
to test our hypothesis, a mkl1-targeting shrna plasmid  or a scrambled control shrna plasmid  were transiently transfected into mpc <dig> cells.
knockdown of mkl <dig> expression was confirmed by western blotting .
compared with shcontrol cells, the cell viability assay indicated that depletion of mkl <dig> promoted mpc <dig> cell proliferation .
the results of dna analysis by flow cytometry further showed that mkl <dig> knockdown mpc <dig> cells had a higher population of cells in s phase and a lower population of cells in g0/g <dig> phase compared with control cells .
the edu cell proliferation assay revealed that repression of mkl <dig> resulted in a significant increase in the number of cells in s phase from  <dig> % to  <dig> % at 72 h after mkl <dig> knockdown .
in addition, mpc <dig> cells were stably transfected with either shmkl <dig>  or shcontrol .
knockdown of mkl <dig> expression was then confirmed by western blotting .
the cell viability and edu cell proliferation assays confirmed that repression of mkl <dig> remarkably promoted cell cycle progression through s phase in mpc <dig> cells .figure  <dig>
knockdown of mkl <dig> promotes cell cycle progression through s phase.
a) mpc <dig> cells were transiently transfected with a mkl1-specific shrna plasmid  or a scrambled shrna control plasmid  and cultured at 33°c.
the efficiency of mkl <dig> protein knockdown was examined by western blotting.
actin was used to normalize mkl <dig> levels.
b) at the indicated time points, cell growth was measured using the cck- <dig> assay.
* p <  <dig>  and **p <  <dig>  compared with the control .
c) cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars, 25 μm.
the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
*p <  <dig>  compared with the control .
d) mpc <dig> cells were stably transfected with a mkl1-specific shrna plasmid  or a scrambled shrna control plasmid  and cultured at 33°c.
expression of mkl <dig> protein was verified by western blotting.
actin was used to normalize mkl <dig> levels.
e) at the indicated time points, cell growth was measured using the cck- <dig> assay.
**p <  <dig>  compared with the control .
f) cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars, 25 μm.
the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
*p <  <dig>  compared with the control .
mkl <dig> regulates podocyte proliferation by targeting p21
to identify the potential cellular pathways regulated by mkl <dig>  differences in the mrna levels of selected signaling molecules were examined using an rt <dig> profiler pcr array by comparing mkl1-expressing mpc <dig> cells with the control cells.
we observed alterations in the expression of several cell cycle regulators, including p <dig>  gadd45a, ddit <dig>  e2f <dig>  and cyclin a <dig> .
qpcr and western blotting were performed to verify these findings .table  <dig>
genes regulated by mkl1
unigene
genebank™ accession no.
symbol
description
fold change
p
valuemkl <dig> induces p <dig> expression.
a) the expression of p <dig> was examined by qpcr and western blotting in mkl1-overexpressing mpc <dig> cells.
gapdh and actin were used to normalize p <dig> levels.
**p <  <dig>  compared with the control .
b) sequential deletions and mutations of the mouse p <dig> promoter were fused to a luciferase reporter.
mpc <dig> cells in 24-well plates were co-transfected with the mkl <dig> expression plasmid  and various wild-type c) or mutant d) p <dig> promoter luciferase reporters .
the luciferase activity of the extracts was determined at  <dig> h after transfection using a betascope analyzer.
luciferase values were normalized to renilla activities.
*p <  <dig>  compared with the empty vector .
e) chip assays were performed using an anti-mkl <dig> antibody, anti-srf antibody, or control igg in mpc <dig> cells.
the association of mkl <dig> or srf with the proximal mouse p <dig> promoter was analyzed by pcr.
the amount of input was confirmed by equal loading of chromatin.
f) chip assays were performed using the anti-mkl <dig> antibody or control igg in mkl1-overexpressing mpc <dig> cells.
the association of mkl <dig> with the proximal mouse p <dig> promoter was analyzed by pcr or qpcr.
the amount of input dna was confirmed by equal loading of chromatin.
*p <  <dig>  compared with the empty vector .
g) Δmkl <dig> cells were transiently transfected with a p21-specific shrna plasmid  and cultured at 33°c.
the efficiency of p <dig> knockdown was examined by western blotting.
actin was used to normalize mkl <dig> and p <dig> levels.
h) cell proliferation was measured by immunofluorescence analysis of edu incorporation.
scale bars,  <dig> μm.
the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%.
*p <  <dig>  compared with the control .
considering that mkl <dig> functions with its co-factor srf by binding to the carg box in the promoter region of target genes  <cit> , we performed a search of the transcription factor database transfac and identified a carg box  at position −316/- <dig> in the mouse p <dig> promoter .
thus, we assessed whether mkl <dig> was a bona fide activator of p <dig> transcription using reporter gene assays.
as shown in figure 4c, mkl <dig> significantly increased mouse p <dig> promoter activity of the wild-type −1562/+ <dig> reporter by approximately 49% relative to the control without mkl <dig> transfection.
furthermore, we found that mkl <dig> activated the promoter activity of p <dig> in a dose-dependent manner .
a series of truncated p <dig> promoter-reporter constructs were thus generated for analysis, as shown in figure 4b.
the results showed that deletion of the carg box significantly abolished mkl1-induced transactivation of the p <dig> promoter compared with that in the control without mkl <dig> transfection .
next, we prepared mutants of the carg box  by site-directed mutagenesis.
we found that mutation of the carg box was sufficient to interfere with mkl1-activated transcription of the p <dig> promoter .
chromatin immunoprecipitation  assays were then performed using an anti-mkl <dig> antibody, anti-srf antibody, or control igg in mpc <dig> cells.
the results indicated that both mkl <dig> and srf were able to bind to the p <dig> promoter during basal conditions in a carg-dependent manner .
overexpression of mkl <dig> resulted in a  <dig> -fold increase in its binding to the endogenous p <dig> promoter in qchip analysis .
these results suggested that the overexpressed mkl <dig> in conjunction with srf promotes p <dig> transcription by binding to the carg box in its promoter.
importantly, to further show that mkl <dig> regulates podocyte proliferation by targeting p <dig>  mkl1-overexpressing mpc <dig> cells  were transfected with a p21-targeting shrna plasmid .
expression of mkl <dig> and p <dig> was assessed by western blotting .
the edu cell proliferation assay revealed a marked decrease in the number of cells in s phase after mkl <dig> overexpression, whereas p <dig> interference remarkably attenuated the mkl1-inhibited cell cycle progression through s phase.
the percentage of cells in s phase increased from  <dig> % to  <dig> % at 48 h after transfection of shp <dig> in mkl1-overexpressing mpc <dig> cells .
these observations confirmed that mkl <dig> inhibits mpc <dig> cell proliferation, which is effectively mediated by targeting p <dig>
the expression of mkl <dig> and p <dig> is positively correlated in podocytes in vivo
next, we detected the appearance of mkl <dig> in developing podocytes by examining its expression in the newborn mouse kidney that displays glomeruli at various stages of development from the s-shaped body through the capillary loop stage to mature glomeruli  <cit> .
immunofluorescence was used to detect mkl <dig> expression in the mouse kidney at postnatal day   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  as seen in figure 5a b-e, s-shaped and comma-shaped bodies were observed in the renal cortex of immature mice at p1– <dig>  ultimately vascularizing into a capillary loop of mature nephron at p <dig>  moreover, mkl <dig> expression was found at all stages of renal glomerulus and tubule formation during postnatal development .
importantly, there was an increase in the expression of mkl <dig> in glomeruli between p <dig> and p <dig> .figure  <dig>
the expression of mkl <dig> and p <dig> is correlated in podocytes
in vivo
.
a) mkl <dig> expression was examined by immunofluorescence staining of the mouse renal cortex at p <dig> , p <dig> , p <dig> , p <dig> , p <dig> , p <dig> , p <dig> , and p <dig> .
the negative control image shows the renal cortex stained with a species-appropriate igg .
scale bar, 50 μm.
b) the expression of p <dig> was examined by immunofluorescent staining in the mouse renal cortex at p <dig> , p <dig> , p <dig> , p <dig> , p <dig>  and p <dig> .
the negative control image was from the renal cortex, in which the primary antibody was a species-appropriate igg .
scale bar, 50 μm.
the s-shaped body , comma-shaped body , capillary loop , and renal tubule  are indicated.
consequently, to address the correlation between mkl <dig> and p <dig> expression in newborn mouse kidney, immunofluorescent staining was used to detect p <dig> expression at p3- <dig>  the results showed a relatively weak expression of p <dig> in glomeruli at p3- <dig> during the postnatal development .
a remarkable upregulation of p <dig> expression was then observed between p <dig> and p <dig> , indicating a positive and sequential correlation between mkl <dig> and p <dig> expression.
considering that podocytes proliferate until the s-shape body stage and exit the cell cycle at the capillary loop stage during glomerulogenesis, these results are consistent with our notion that mkl <dig> functions as an effective inducer of cell growth arrest in podocytes, that might be mediated by the regulation of p <dig>
discussion
growth arrest and differentiation of podocytes are essential for normal formation of glomeruli in the developing kidney and paramount for normal glomerular function in the mature kidney.
the precise cell cycle regulation necessary to establish podocyte quiescence during development has not been fully defined.
in the present study, we identified the contribution of one of the myocardin/mkl proteins, mkl <dig>  to the regulation of mpc <dig> cell proliferation.
during temperature-switched growth arrest of mpc <dig> cells, mkl <dig> expression was significantly upregulated above its other family members.
consequently, experiments were performed to assess gain- and loss-of-function of mkl <dig> to study the effect of mkl <dig> on mpc <dig> cell proliferation.
we found that overexpression of mkl <dig> resulted in significant repression of g1/s phase progression of the cell cycle, whereas mkl <dig> knockdown had the opposite effect.
importantly, we demonstrated that mkl <dig> activated transcription of the cell cycle inhibitor p <dig> by recruitment to its promoter in a carg element-dependent manner.
in addition, mkl <dig> expression in the mouse kidney during postnatal development showed that mkl <dig> is expressed in podocytes in vivo, which was significantly upregulated during the morphological switch of podocytes from proliferation to differentiation.
recent evidence supports an important and complex role of mkl <dig> in maintenance of proper differentiation in various cell lines including smooth muscle cells  <cit> , myofibroblasts  <cit> , megakaryocytes  <cit> , and adipocytes  <cit> .
whether mkl <dig> functions in cell proliferation, however, remains poorly understood.
shaposhnikov et al.
reported that mkl <dig> regulates the expression levels of two proapoptotic bcl- <dig> family members, bok and noxa, and thus is involved in apoptotic signaling in nih3t <dig> fibroblasts  <cit> .
here, we extended the study to show that elevated expression of mkl <dig> significantly blocked cell cycle progression by decreasing the number of mpc <dig> cells in s phase.
on the other hand, rna interference of mkl <dig> expression resulted in the opposite effect to promote g1/s transition of the cell cycle, confirming a potential role of mkl <dig> in regulation of cell proliferation.
these observations collectively position mkl <dig> as a potential regulatory factor at the crossroad between cell proliferation and differentiation.
indeed, myocardin family members have been previously implicated in the regulation of cell proliferation.
myocardin impairs the proliferation of vascular smooth muscle cells, chinese hamster ovarian  cells, and leiomyosarcoma cells  <cit> .
in ht <dig> fibrosarcoma cells, myocardin inhibits proliferation at a low cell density and abrogates colony formation in soft agar  <cit> .
however, the suggested molecular mechanisms underlying myocardin/mkl-induced cell growth arrest are conflicting.
in human uterine leiomyosarcoma cells, myocardin in conjunction with srf directly binds to the p <dig> promoter and induces its expression, thus resulting in g1/s arrest  <cit> .
in contrast, myocardin has been shown to downregulate expression of c-myc, cdk <dig>  cdk <dig>  and s6k, but not p <dig> and p <dig>  which leads to g2/m arrest and accumulation of polyploidy cells  <cit> .
our data presented here support the notion that mkl <dig> functions as an effective regulator to inhibit cell proliferation by altering p <dig> expression in mpc <dig> cells.
significant upregulation of p <dig> expression at both mrna and protein levels was observed after transfecting the mkl <dig> expression plasmid, whereas mkl <dig> interference resulted in the opposite effect to downregulate p <dig> expression , suggesting that a direct mechanism is involved in this regulation.
we further demonstrated that mkl <dig> induced promoter activity of the p <dig> gene in a dose-dependent manner.
importantly, we found that deletion or mutation of the carg element in the mouse p <dig> promoter remarkably abolished the stimulatory effect on p <dig> transcription induced by mkl <dig>  transfection of the mkl <dig> expression plasmid led to a marked increase in the binding affinity of mkl <dig> for the endogenous p <dig> promoter, indicating a significant role of the carg element in mediating mkl1-induced expression of p <dig>  in addition to p <dig>  we identified obvious candidates involved in mkl1-regulated mpc <dig> cell proliferation, such as gadd45a, ddit <dig>  e2f <dig>  and cyclin a <dig>  however, these genes are not potential targets of myocardin/mkls/srf .
these results indicate that an srf-independent mechanism might contribute to mkl-mediated g1/s arrest of the cell cycle.
in the present study, we found that mkl <dig> was expressed in podocytes of the mouse kidney during postnatal development.
moreover, a significant increase in mkl <dig> expression was observed between p <dig> and p <dig> during postnatal development of the kidney, highlighting a potential role of mkl <dig> in the physiological and morphological switch of podocytes from proliferation to differentiation.
therefore, using the conditionally immortalized mouse podocyte cell line mpc <dig>  we further revealed that mkl <dig> functioned as an effective inducer to inhibit cell proliferation and trigger cell cycle arrest at g1/s transition.
several studies have also demonstrated the presence of an intrinsic barrier to replication associated with activation of the cell cycle in podocytes.
re-expression of cell cycle proteins has been reported during glomerular disorders.
de novo cyclin a staining is observed in podocytes of children with collapsing glomerulopathy  <cit>  and focal segmental glomerulosclerosis   <cit> .
positive signals for cyclin d have also been reported in the cellular lesions of fsgs  <cit> .
recently, strong upregulation of ckis p <dig> and p <dig> was reported in podocytes during heymann nephritis and in diabetic zdf-fa/fa rats  <cit> .
moreover, the glomerular tufts in crescentic glomerulonephritis strongly express ckis  <cit> , suggesting that podocytes upregulate ckis to maintain cell cycle quiescence and preserve normal physiological functions.
here, we extended the study showing that mkl <dig> acted as an upstream regulator of a variety of cell cycle factors, such as p <dig> and cyclin a <dig>  to control cell cycle progression in podocytes.
in addition, we found significant upregulation of mkl <dig> expression in the renal tubular cells of newborn mouse kidneys.
recent reports have revealed a potential role of mkl <dig> in the regulation of renal tubular diseases.
for example, xu et al.
reported that mkl <dig> is induced by glucose and synergizes with glucose to induce collagen expression in cultured renal tubular epithelial cells and the kidneys of mice with diabetic nephropathy, eventually leading to tubulointerstitial fibrosis  <cit> .
moreover, suppressor of cancer cell invasion  has been demonstrated to negatively regulate epithelial-mesenchymal transition and renal fibrosis in llc-pk <dig>  proximal tubular epithelial cells, which is at least partially mediated by repression of mkl <dig> and mkl <dig>  <cit> .
taken together, these observations indicate that mkl <dig> performs physiological roles in maturation and development of the kidney, and thus its dysfunction might lead to glomerular and renal diseases.
CONCLUSIONS
in the present study, we found a potential mechanism of mkl1/p21-mediated cell cycle quiescence in podocytes.
therefore, these findings reveal a novel function of mkl <dig> during podocyte proliferation and differentiation, furthering our understanding of kidney development and the mechanisms of kidney diseases.

 podocytes, also called visceral glomerular epithelial cells, are terminally differentiated cells overlaying the outer region of the glomerular basement membrane of renal glomeruli. these cells have several key functions including the prevention of proteinuria, synthesis of basement membrane components, regulation of glomerular filtration, and counteraction of intraglomerular hydrostatic pressure. podocyte injury is typically associated with proteinuria and progressive glomerulosclerosis. podocytes are derived from epithelial cells originating in the metanephric mesenchyme, which develop into postmitotic terminally differentiated cells, and therefore have similarities to neurons. during glomerulogenesis, podocytes proliferate until the s-shape body stage and exit the cell cycle at the capillary loop stage. podocytes then acquire their fully differentiated phenotype, a process that is not complete until 1 week after birth in the mouse. mature podocytes tightly regulate and maintain their quiescent and differentiated phenotype, and therefore lost podocytes cannot be replaced by proliferation of neighboring undamaged cells. indeed, studies have shown that the inability to proliferate contributes to glomerular scarring. thus, the mechanism responsible for the cell cycle arrest that occurs during nephrogenesis may also participate in maintenance of cell cycle quiescence in mature podocytes. megakaryoblastic leukemia was originally found in a study of a chromosomal translocation, t, which is closely related to the incidence of acute megakaryoblastic leukemia in infants and children. mkl has been recently shown to be a member of a three-protein family that includes mkl and myocardin. these myocardin/mkl proteins serve as serum response factor coactivators by binding to srf and strongly activating srf target genes. in contrast to myocardin, which has cardiac and smooth muscle-specific expression, mkl and mkl are expressed in a wide range of embryonic and adult tissues. mkl regulates many processes including muscle cell differentiation, cardiovascular development, remodeling of neuronal networks in the developing and adult brain, megakaryocytic differentiation and migration, modulation of cellular motile functions, and epithelial-mesenchymal transition. notably, there is increasing evidence of the involvement of the myocardin/mkl family in suppression of cell proliferation and cell cycle progression. both myocardin and mkl exert anti-proliferative effects in various cell lines. therefore, unraveling the functional pathways in which these proteins have a role and furthering our comprehension of the cellular mechanisms intrinsic to their regulation of cell proliferation will become increasingly important. in the present study, we found that mkl expression was upregulated during temperature-switched growth arrest in murine podocyte clone cells. overexpression of mkl resulted in inhibition of g1/s cell cycle progression in cell viability and edu cell proliferation assays, whereas mkl knockdown had the opposite effect. we further demonstrated that mkl induced expression of the cyclin-dependent kinase inhibitor p during the regulation of cell cycle arrest. importantly, mkl expression was observed in podocytes of the mouse kidney during postnatal development, which was upregulated during the morphological switch of podocytes from proliferation to differentiation.  expression of mkl is upregulated during temperature-switched cell growth arrest in mpc cells to assess the possible role of myocardin/mkl proteins in podocyte growth arrest, mpc cells were respectively maintained at the permissive temperature of 33°c and at the nonpermissive temperature of 37°c for 10 days. as shown in figure 1a, a cell viability assay indicated that mpc cells cultured at 37°c showed a significant decrease in cell number compared with those cultured at 33°c. the results of immunofluorescence staining in the edu cell proliferation assay further revealed a marked decrease in the number of cells in s phase by the temperature switch to 37°c. the percentage of cells in s phase decreased from % to % as early as 2 days after the temperature switch. at 4– days, the percentage of cells in s phase further decreased from % to %, which is consistent with a previous report indicating that the temperature switch induces growth arrest of podocytes in vitro.figure mkl is upregulated during temperature-switched cell cycle arrest in mpc cells. a) mpc cells were cultured at the permissive temperature of 33°c or the nonpermissive temperature of 37°c. at the indicated time points, cell growth was measured using a cck- assay. **p <  compared with the control. b) mpc cells were shifted from 33°c to 37°c and cultured for the indicated times. cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, 25 μm. c) the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. **p <  compared with the control. d) mpc cells were shifted from 33°c to 37°c and cultured for the indicated times. the mrna expression levels of myocardin, mkl and mkl were verified by qpcr. gapdh was used to normalize expression levels. *p <  compared with the control. e) mpc cells were shifted from 33°c to 37°c and cultured for the indicated times. protein expression levels of mkl were examined by western blotting. actin was used to normalize mkl levels. the expression of myocardin/mkl proteins was then measured during temperature-switched growth arrest in mpc cells. qpcr analysis indicated that the temperature switch to 37°c induced an approximate -fold increase in mkl mrna expression compared with the basal level at 2 days. at 4– days, mkl expression showed a 2-4-fold increase at the mrna level. western blotting was used to confirm the upregulation of mkl expression at the protein level. however, the alteration in myocardin and mkl expression was not as evident. considering the dominant presence of mkl over its other family members, we focused on the effects of mkl in subsequent experiments. mkl functions as an effective inducer of cell growth arrest in mpc cells next, a mouse mkl expression plasmid was transiently transfected into mpc cells. overexpression of mkl was assessed by western blotting. compared with control cells, the cell viability assay indicated that ectopic expression of mkl inhibited mpc cell proliferation. results of dna analysis by flow cytometry further confirmed that mkl1-overexpressing mpc cells had a lower population of s phase cells and a higher population of g0/g phase cells. the edu cell proliferation assay revealed a marked decrease in the number of s phase cells after mkl overexpression. the percentage of cells in s phase decreased from % to % at 72 h after transfection of the mkl expression plasmid. furthermore, mpc cells were stably transfected with either the mkl expression plasmid or the empty vector. overexpression of mkl was then examined by western blotting. cell viability and edu cell proliferation assays confirmed that mkl overexpression induced a delay in g1/s phase transition of mpc cells.figure overexpression of mkl induces mpc cell growth arrest. a) mpc cells were transiently transfected with a mouse mkl expression plasmid and cultured at 33°c. expression of mkl protein was verified by western blotting. actin was used to normalize mkl levels. b) at the indicated time points, cell growth was measured using the cck- assay. *p <  and **p <  compared with the control. c) cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, 25 μm. the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. *p <  compared with the control. d) mpc cells were stably transfected with a mouse mkl expression plasmid and cultured at 33°c. expression of mkl protein was verified by western blotting. actin was used to normalize mkl levels. e) at the indicated time points, cell growth was measured using the cck- assay. **p <  compared with the control. f) cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, 25 μm. the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. *p <  compared with the control. therefore, we hypothesized that knockdown of mkl by rna interference would result in an increase in the number of cells in s phase. to test our hypothesis, a mkl1-targeting shrna plasmid or a scrambled control shrna plasmid were transiently transfected into mpc cells. knockdown of mkl expression was confirmed by western blotting. compared with shcontrol cells, the cell viability assay indicated that depletion of mkl promoted mpc cell proliferation. the results of dna analysis by flow cytometry further showed that mkl knockdown mpc cells had a higher population of cells in s phase and a lower population of cells in g0/g phase compared with control cells. the edu cell proliferation assay revealed that repression of mkl resulted in a significant increase in the number of cells in s phase from % to % at 72 h after mkl knockdown. in addition, mpc cells were stably transfected with either shmkl or shcontrol. knockdown of mkl expression was then confirmed by western blotting. the cell viability and edu cell proliferation assays confirmed that repression of mkl remarkably promoted cell cycle progression through s phase in mpc cells.figure knockdown of mkl promotes cell cycle progression through s phase. a) mpc cells were transiently transfected with a mkl1-specific shrna plasmid or a scrambled shrna control plasmid and cultured at 33°c. the efficiency of mkl protein knockdown was examined by western blotting. actin was used to normalize mkl levels. b) at the indicated time points, cell growth was measured using the cck- assay. * p <  and **p <  compared with the control. c) cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, 25 μm. the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. *p <  compared with the control. d) mpc cells were stably transfected with a mkl1-specific shrna plasmid or a scrambled shrna control plasmid and cultured at 33°c. expression of mkl protein was verified by western blotting. actin was used to normalize mkl levels. e) at the indicated time points, cell growth was measured using the cck- assay. **p <  compared with the control. f) cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, 25 μm. the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. *p <  compared with the control. mkl regulates podocyte proliferation by targeting p21 to identify the potential cellular pathways regulated by mkl differences in the mrna levels of selected signaling molecules were examined using an rt profiler pcr array by comparing mkl1-expressing mpc cells with the control cells. we observed alterations in the expression of several cell cycle regulators, including p gadd45a, ddit e2f and cyclin a. qpcr and western blotting were performed to verify these findings.table genes regulated by mkl1 unigene genebank™ accession no. symbol description fold change p valuemkl induces p expression. a) the expression of p was examined by qpcr and western blotting in mkl1-overexpressing mpc cells. gapdh and actin were used to normalize p levels. **p < compared with the control. b) sequential deletions and mutations of the mouse p promoter were fused to a luciferase reporter. mpc cells in 24-well plates were co-transfected with the mkl expression plasmid and various wild-type c) or mutant d) p promoter luciferase reporters. the luciferase activity of the extracts was determined at h after transfection using a betascope analyzer. luciferase values were normalized to renilla activities. *p < compared with the empty vector. e) chip assays were performed using an anti-mkl antibody, anti-srf antibody, or control igg in mpc cells. the association of mkl or srf with the proximal mouse p promoter was analyzed by pcr. the amount of input was confirmed by equal loading of chromatin. f) chip assays were performed using the anti-mkl antibody or control igg in mkl1-overexpressing mpc cells. the association of mkl with the proximal mouse p promoter was analyzed by pcr or qpcr. the amount of input dna was confirmed by equal loading of chromatin. *p < compared with the empty vector. g) Δmkl cells were transiently transfected with a p21-specific shrna plasmid and cultured at 33°c. the efficiency of p knockdown was examined by western blotting. actin was used to normalize mkl and p levels. h) cell proliferation was measured by immunofluorescence analysis of edu incorporation. scale bars, μm. the percentage of proliferating cells was calculated as edu-positive cells/hoechst-stained cells × 100%. *p < compared with the control. considering that mkl functions with its co-factor srf by binding to the carg box in the promoter region of target genes, we performed a search of the transcription factor database transfac and identified a carg box at position −316/- in the mouse p promoter. thus, we assessed whether mkl was a bona fide activator of p transcription using reporter gene assays. as shown in figure 4c, mkl significantly increased mouse p promoter activity of the wild-type −1562/+ reporter by approximately 49% relative to the control without mkl transfection. furthermore, we found that mkl activated the promoter activity of p in a dose-dependent manner. a series of truncated p promoter-reporter constructs were thus generated for analysis, as shown in figure 4b. the results showed that deletion of the carg box significantly abolished mkl1-induced transactivation of the p promoter compared with that in the control without mkl transfection. next, we prepared mutants of the carg box by site-directed mutagenesis. we found that mutation of the carg box was sufficient to interfere with mkl1-activated transcription of the p promoter. chromatin immunoprecipitation assays were then performed using an anti-mkl antibody, anti-srf antibody, or control igg in mpc cells. the results indicated that both mkl and srf were able to bind to the p promoter during basal conditions in a carg-dependent manner. overexpression of mkl resulted in a -fold increase in its binding to the endogenous p promoter in qchip analysis. these results suggested that the overexpressed mkl in conjunction with srf promotes p transcription by binding to the carg box in its promoter. importantly, to further show that mkl regulates podocyte proliferation by targeting p mkl1-overexpressing mpc cells were transfected with a p21-targeting shrna plasmid. expression of mkl and p was assessed by western blotting. the edu cell proliferation assay revealed a marked decrease in the number of cells in s phase after mkl overexpression, whereas p interference remarkably attenuated the mkl1-inhibited cell cycle progression through s phase. the percentage of cells in s phase increased from % to % at 48 h after transfection of shp in mkl1-overexpressing mpc cells. these observations confirmed that mkl inhibits mpc cell proliferation, which is effectively mediated by targeting p the expression of mkl and p is positively correlated in podocytes in vivo next, we detected the appearance of mkl in developing podocytes by examining its expression in the newborn mouse kidney that displays glomeruli at various stages of development from the s-shaped body through the capillary loop stage to mature glomeruli. immunofluorescence was used to detect mkl expression in the mouse kidney at postnatal day and as seen in figure 5a b-e, s-shaped and comma-shaped bodies were observed in the renal cortex of immature mice at p1– ultimately vascularizing into a capillary loop of mature nephron at p moreover, mkl expression was found at all stages of renal glomerulus and tubule formation during postnatal development. importantly, there was an increase in the expression of mkl in glomeruli between p and p.figure the expression of mkl and p is correlated in podocytes in vivo. a) mkl expression was examined by immunofluorescence staining of the mouse renal cortex at p, p, p, p, p, p, p, and p. the negative control image shows the renal cortex stained with a species-appropriate igg. scale bar, 50 μm. b) the expression of p was examined by immunofluorescent staining in the mouse renal cortex at p, p, p, p, p and p. the negative control image was from the renal cortex, in which the primary antibody was a species-appropriate igg. scale bar, 50 μm. the s-shaped body, comma-shaped body, capillary loop, and renal tubule are indicated. consequently, to address the correlation between mkl and p expression in newborn mouse kidney, immunofluorescent staining was used to detect p expression at p3- the results showed a relatively weak expression of p in glomeruli at p3- during the postnatal development. a remarkable upregulation of p expression was then observed between p and p, indicating a positive and sequential correlation between mkl and p expression. considering that podocytes proliferate until the s-shape body stage and exit the cell cycle at the capillary loop stage during glomerulogenesis, these results are consistent with our notion that mkl functions as an effective inducer of cell growth arrest in podocytes, that might be mediated by the regulation of p discussion growth arrest and differentiation of podocytes are essential for normal formation of glomeruli in the developing kidney and paramount for normal glomerular function in the mature kidney. the precise cell cycle regulation necessary to establish podocyte quiescence during development has not been fully defined. in the present study, we identified the contribution of one of the myocardin/mkl proteins, mkl to the regulation of mpc cell proliferation. during temperature-switched growth arrest of mpc cells, mkl expression was significantly upregulated above its other family members. consequently, experiments were performed to assess gain- and loss-of-function of mkl to study the effect of mkl on mpc cell proliferation. we found that overexpression of mkl resulted in significant repression of g1/s phase progression of the cell cycle, whereas mkl knockdown had the opposite effect. importantly, we demonstrated that mkl activated transcription of the cell cycle inhibitor p by recruitment to its promoter in a carg element-dependent manner. in addition, mkl expression in the mouse kidney during postnatal development showed that mkl is expressed in podocytes in vivo, which was significantly upregulated during the morphological switch of podocytes from proliferation to differentiation. recent evidence supports an important and complex role of mkl in maintenance of proper differentiation in various cell lines including smooth muscle cells, myofibroblasts, megakaryocytes, and adipocytes. whether mkl functions in cell proliferation, however, remains poorly understood. shaposhnikov et al. reported that mkl regulates the expression levels of two proapoptotic bcl- family members, bok and noxa, and thus is involved in apoptotic signaling in nih3t fibroblasts. here, we extended the study to show that elevated expression of mkl significantly blocked cell cycle progression by decreasing the number of mpc cells in s phase. on the other hand, rna interference of mkl expression resulted in the opposite effect to promote g1/s transition of the cell cycle, confirming a potential role of mkl in regulation of cell proliferation. these observations collectively position mkl as a potential regulatory factor at the crossroad between cell proliferation and differentiation. indeed, myocardin family members have been previously implicated in the regulation of cell proliferation. myocardin impairs the proliferation of vascular smooth muscle cells, chinese hamster ovarian cells, and leiomyosarcoma cells. in ht fibrosarcoma cells, myocardin inhibits proliferation at a low cell density and abrogates colony formation in soft agar. however, the suggested molecular mechanisms underlying myocardin/mkl-induced cell growth arrest are conflicting. in human uterine leiomyosarcoma cells, myocardin in conjunction with srf directly binds to the p promoter and induces its expression, thus resulting in g1/s arrest. in contrast, myocardin has been shown to downregulate expression of c-myc, cdk cdk and s6k, but not p and p which leads to g2/m arrest and accumulation of polyploidy cells. our data presented here support the notion that mkl functions as an effective regulator to inhibit cell proliferation by altering p expression in mpc cells. significant upregulation of p expression at both mrna and protein levels was observed after transfecting the mkl expression plasmid, whereas mkl interference resulted in the opposite effect to downregulate p expression, suggesting that a direct mechanism is involved in this regulation. we further demonstrated that mkl induced promoter activity of the p gene in a dose-dependent manner. importantly, we found that deletion or mutation of the carg element in the mouse p promoter remarkably abolished the stimulatory effect on p transcription induced by mkl transfection of the mkl expression plasmid led to a marked increase in the binding affinity of mkl for the endogenous p promoter, indicating a significant role of the carg element in mediating mkl1-induced expression of p in addition to p we identified obvious candidates involved in mkl1-regulated mpc cell proliferation, such as gadd45a, ddit e2f and cyclin a however, these genes are not potential targets of myocardin/mkls/srf. these results indicate that an srf-independent mechanism might contribute to mkl-mediated g1/s arrest of the cell cycle. in the present study, we found that mkl was expressed in podocytes of the mouse kidney during postnatal development. moreover, a significant increase in mkl expression was observed between p and p during postnatal development of the kidney, highlighting a potential role of mkl in the physiological and morphological switch of podocytes from proliferation to differentiation. therefore, using the conditionally immortalized mouse podocyte cell line mpc we further revealed that mkl functioned as an effective inducer to inhibit cell proliferation and trigger cell cycle arrest at g1/s transition. several studies have also demonstrated the presence of an intrinsic barrier to replication associated with activation of the cell cycle in podocytes. re-expression of cell cycle proteins has been reported during glomerular disorders. de novo cyclin a staining is observed in podocytes of children with collapsing glomerulopathy and focal segmental glomerulosclerosis. positive signals for cyclin d have also been reported in the cellular lesions of fsgs. recently, strong upregulation of ckis p and p was reported in podocytes during heymann nephritis and in diabetic zdf-fa/fa rats. moreover, the glomerular tufts in crescentic glomerulonephritis strongly express ckis, suggesting that podocytes upregulate ckis to maintain cell cycle quiescence and preserve normal physiological functions. here, we extended the study showing that mkl acted as an upstream regulator of a variety of cell cycle factors, such as p and cyclin a to control cell cycle progression in podocytes. in addition, we found significant upregulation of mkl expression in the renal tubular cells of newborn mouse kidneys. recent reports have revealed a potential role of mkl in the regulation of renal tubular diseases. for example, xu et al. reported that mkl is induced by glucose and synergizes with glucose to induce collagen expression in cultured renal tubular epithelial cells and the kidneys of mice with diabetic nephropathy, eventually leading to tubulointerstitial fibrosis. moreover, suppressor of cancer cell invasion has been demonstrated to negatively regulate epithelial-mesenchymal transition and renal fibrosis in llc-pk proximal tubular epithelial cells, which is at least partially mediated by repression of mkl and mkl. taken together, these observations indicate that mkl performs physiological roles in maturation and development of the kidney, and thus its dysfunction might lead to glomerular and renal diseases.  in the present study, we found a potential mechanism of mkl1/p21-mediated cell cycle quiescence in podocytes. therefore, these findings reveal a novel function of mkl during podocyte proliferation and differentiation, furthering our understanding of kidney development and the mechanisms of kidney diseases. 
in the current study, we found that the expression of a myocardin/mkl family member, mkl was significantly upregulated during cell cycle arrest induced by a temperature switch in murine podocyte clone cells. in contrast, mkl knockdown by rna interference had the opposite effect, highlighting a potential role of mkl in blocking g1/s transition of the cell cycle in mpc cells. additionally, using an rt profiler pcr array, p was identified as a direct target of mkl we further revealed that mkl activated p transcription by recruitment to the carg element in its promoter, thus resulting in cell cycle arrest. further investigation demonstrated that overexpression of mkl led to inhibition of cell proliferation by decreasing the number of cells in s phase of the cell cycle. in addition, the expression of mkl is positively correlated with that of p in podocytes in postnatal mouse kidney and significantly upregulated during the morphological switch of podocytes from proliferation to differentiation.
 the glomerular podocyte is a highly specialized cell type with the ability to ultrafilter blood and support glomerular capillary pressure. however, little is known about the genetic programs leading to this functionality or the final phenotype.  in the current study, we found that the expression of a myocardin/mkl family member, mkl was significantly upregulated during cell cycle arrest induced by a temperature switch in murine podocyte clone cells. further investigation demonstrated that overexpression of mkl led to inhibition of cell proliferation by decreasing the number of cells in s phase of the cell cycle. in contrast, mkl knockdown by rna interference had the opposite effect, highlighting a potential role of mkl in blocking g1/s transition of the cell cycle in mpc cells. additionally, using an rt profiler pcr array, p was identified as a direct target of mkl we further revealed that mkl activated p transcription by recruitment to the carg element in its promoter, thus resulting in cell cycle arrest. in addition, the expression of mkl is positively correlated with that of p in podocytes in postnatal mouse kidney and significantly upregulated during the morphological switch of podocytes from proliferation to differentiation.  our observations demonstrate that mkl has physiological roles in the maturation and development of podocytes, and thus its misregulation might lead to glomerular and renal dysfunction. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords kidney developmentpodocytecell growth arrestmkl1issue-copyright-statement© the author 2015 