The Smad signalling pathway is critical for transmitting transforming growth factor-beta
(TGF-beta) superfamily signals from the cell surface to the nucleus. In the nucleus,
Smads regulate transcriptional responses by recruiting co-activators and co-repressors
to a wide array of DNA-binding partners. Thus, Smads function as transcriptional
co-modulators to regulate TGFbeta-dependent gene expression. Derynck et al. (1996) proposed a revised nomenclature for the Mad-related products and genes that are
implicated in signal transduction by members of the TGF-beta family. As the root symbol they proposed SMAD, which is a
merger of Sma (the gene in C. elegans) and Mad. SMAD serves to differentiate these proteins from unrelated gene products
previously called MAD.
NCBI Summary:
The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to TGF-beta signal, this protein is phosphorylated by the TGF-beta receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The association with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, May 2012]
General function
Intracellular signaling cascade
Comment
Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality. Kuo FT et al. PURPOSE: To determine the expression of SMAD transcripts in human granulosa cells. METHODS: Luteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR). RESULTS: SMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p?=?0.0062) and trended with SMAD4:SMAD2 (p?=?0.0698). CONCLUSIONS: SMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.
Cellular localization
Cytoplasmic, Nuclear
Comment
Ovarian function
Initiation of primordial follicle growth, Cumulus expansion, Ovulation, Oogenesis
Comment
Nuclear exclusion of SMAD2/3 in granulosa cells is associated with primordial follicle activation in the mouse ovary. Hardy K et al. (2018) Maintenance and activation of the limited supply of primordial follicles in the ovary are important determinants of reproductive lifespan. Currently, the molecular programme that maintains the primordial phenotype and the early events associated with follicle activation are not well defined. Here we have systematically analysed these events using microscopy and detailed image analysis. Using the immature mouse ovary as a model, we demonstrate that the onset of granulosa cell (GC) proliferation results in increased packing density on the oocyte surface and consequent GC cuboidalisation. These events precede oocyte growth and nuclear translocation of FOXO3a, a transcription factor important in follicle activation. Immunolabelling of the TGFβ signalling mediators and transcription factors, SMAD2/3, revealed a striking expression pattern specific to GCs of small follicles. SMAD2/3 was expressed in the nuclei of primordial GCs but was mostly excluded in early growing follicles. In activated follicles, GC nuclei lacking SMAD2/3 generally expressed Ki67. These findings suggest that the first phenotypic changes during follicle activation are observed in GCs, and that TGFβ signalling is fundamental for regulating GC arrest and the onset of proliferation.//////////////////
Oocytes determine cumulus cell lineage in mouse ovarian follicles. Diaz FJ et al. The two principal functions of ovarian follicles are developmental and endocrine. The cumulus cells surrounding the oocyte are specialized to serve the development of the oocyte and steroidogenesis is a principal role of mural granulosa cells that line the follicle wall. The findings in this report demonstrate that oocytectomy or treatment with an inhibitor of SMAD2/3 activation results in decreased cumulus marker mRNA transcript levels and allows FSH to induce mural marker transcripts in cumulus cells. In addition, SMAD2/3 signaling is involved in enabling cumulus expansion and EGF-induced increases in Ptx3, Ptgs2 and Has2 mRNA levels. By contrast, follicle-stimulating hormone (FSH) stimulated expression of mural transcripts, but suppressed levels of cumulus transcripts. Thus, FSH and oocyte-stimulated SMAD2/3 signaling establish opposing gradients of influence in the follicle. These specify the mural and cumulus granulosa cell phenotypes that are pivotal for appropriate endocrine function and oocyte development.
Expression regulated by
FSH, Growth Factors/ cytokines, mir4110
Comment
Chi-miR-4110 promotes granulosa cell apoptosis by targeting Sma- and Mad-related protein 2 (Smad2) in the caprine ovary. An X et al. (2017) Follicular atresia mainly results from the apoptosis of granulosa cells (GCs). Whilst our previous investigations examined the role of chi-miR-4110 in regulating ovarian function, the present study detected the role of chi-miR-4110 in GC development. We transfected caprine GCs cultured in vitro with chi-miR-4110 mimics. Results revealed that chi-miR-4110 decreased mRNA and protein levels of Smad2 by targeting its 3'-untranslated region (3'UTR). FoxC1 and Sp1 mRNA and protein levels markedly increased, whereas those of bHLHe22 significantly decreased (P<0.01 or 0.05) in GCs transfected with the chi-miR-4110 mimics. Further studies revealed a significantly higher number of apoptotic cells in GCs transfected with the chi-miR-4110 mimics (P< 0.05) than in GCs transfected with mimics negative control. GCs transfected with the chi-miR-4110 mimics exhibited significantly increased mRNA and protein levels of the pro-apoptotic gene Bax (P<0.01) and significantly decreased expression levels of the anti-apoptotic gene BCL-2 (P<0.01). Smad2 interference (Si-1282) results were consistent with those of the chi-miR-4110 mimics. Previous reports and our results showed that chi-miR-4110 increases Sp1 expression by repressing Smad2. The increase in Sp1 induces p53-upregulated modulator of apoptosis, which increases the relative abundance of Bax and causes caprine GC apoptosis. Our findings may provide relevant data for the investigation of miRNA-mediated regulation of ovarian functions.//////////////////
Li M, et al reported that Mothers against decapentaplegic-related protein 2 expression in
avian granulosa cells is up-regulated by transforming growth
factor beta during ovarian follicular development.
They demonstrated the presence of MADR2 and
MADR1 in hen granulosa cells at different stages of follicular development. The
expression of MADR2, but not of MADR1, was up-regulated by TGFbeta in vitro
in a concentration- and time-dependent manner. Granulosa cell MADR2
expression was maximal during early stages of follicular development.
Schmierer B,et al 2003 reported that activin A signaling induces Smad2, but not Smad3, requiring protein kinase A
activity in granulosa cells from the avian ovary.
Activin A signaling is an important regulator of ovarian granulosa cell function. The cytosolic signal
transducer Smad2 is most highly expressed in chicken granulosa cells (cGC) of pre-ovulatory
follicles (POF's). Moreover, Smad2 shows predominant nuclear localization in freshly isolated
cGC, indicating active Smad-signaling in vivo. Primary cGC cultured in vitro require activin A to
sustain high Smad2 levels, which otherwise drop dramatically in the absence of activin A. This
activin A-dependent Smad2 expression is abrogated by protein kinase A (PKA) inhibitors,
suggesting a role for PKA in activin signaling. In the absence of activin A, strong PKA activators
such as follicle-stimulating hormone (FSH) and 8-Br-cAMP fail to elicit Smad2 induction.
However, FSH and 8-Br-cAMP boost activin A-dependent Smad2 upregulation, giving rise to
Smad2 levels similar to expression in vivo levels. Interestingly, the effect is specific for Smad2, as
expression of the structurally and functionally closely related Smad3 remains entirely unaffected.
Hence, activin A induces Smad2, but not Smad3, to high levels requiring PKA activation. As Smad2
and Smad3 target distinct, yet overlapping sets of TGF-beta/activin responsive genes, the selective
Smad2 induction by FSH/activin A could allow FSH to efficiently modulate the transcriptional
readout of activin A signaling in avian granulosa cells.
Ovarian localization
Oocyte, Granulosa, Theca
Comment
Expression and localization of Smad2 and Smad4 proteins in the porcine ovary. Xing N 2014 et al.
The objective of the present study was to investigate the temporal and spatial expression of Smad2 and Smad4 proteins, the downstream signaling molecules of the transforming growth factor beta (TGF-) superfamily, in the porcine ovary. Cellular localization of Smad2 and Smad4 proteins was examined using immunohistochemistry. The specificity of the antibodies was examined using Western blot assay. Western blot analyses demonstrated that 52kDa Smad2 and 60kDa Smad4 proteins were expressed in the porcine ovary. Immunohistochemistry revealed that Smad2 and Smad4 were widely expressed in the porcine ovary, mainly localized in the oocyte, granulosa and thecal cells at different stages of folliculogenesis. Within the primordial and primary follicles, Smad2 and Smad4 showed strong staining in oocytes and follicular cells. In the antral follicle, strong staining was observed in oocytes, granulosa and theca cells. These findings suggest that Smad2 and Smad4 may be a key regulator of follicular development and growth of oocytes in the porcine ovary.
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?terlund et al 2000
used reverse transcription-polymerase chain reaction (RT-PCR) and
immunohistochemistry to investigate the presence in human oocytes and preimplantation embryos of the essential components
of the TGF?signalling pathway, TGF?receptors type I and II and the substrate proteins Smad 2 and 3. It was found that both
receptors, as well as Smad 2 and 3, were present in the unfertilized oocyte, whereas only the type I receptor and Smad 2 and
3 were present at the blastocyst stage. At the 4-cell and 8-cell stages neither of the receptors was present, but Smad 2 and 3
were present at both stages.
Billiar RB, et al determined whether the primate fetal ovary expressed Smads 2/3 and 4 and whether expression of these activin-signaling proteins was altered in fetal ovaries of baboons in which estrogen production was suppressed. Western blot analyses demonstrated that the 59 kDa Smad 2, 54 kDa Smad 3, and 64 kDa Smad 4 proteins were expressed in fetal ovaries of untreated baboons at both mid and late gestation and that the level of expression was not significantly altered in late gestation by in vivo treatment with CGS 20267 or CGS 20267 and estrogen. Immunocytochemistry localized Smads 2/3 and 4 to cytoplasm of oocytes and pregranulosa cells at midgestation and oocytes and granulosa cells of primordial follicles in late gestation. Smad 4 was also detected in granulosa cell nuclei in late gestation and nuclear expression appeared to be decreased in fetal ovaries of baboons deprived of estrogen. The site of localization of Smads correlated with localization of the activin receptors IA and IIB which we previously showed were abundantly expressed in oocytes and (pre)granulosa cells at both mid and late gestation and unaltered by estrogen deprivation. In summary, the results of the current study are the first to show that the intracellular signaling molecules required to transduce an activin signal are expressed in the baboon fetal ovary and that expression was not altered by estrogen-deprivation in utero. These findings coupled with our previous observations showing that estrogen-deprivation reduced follicle numbers and up-regulated/induced expression of inhibin but not activin or the activin receptors, lend further support to the hypothesis that estrogen regulates fetal ovarian folliculogenesis by controlling the intraovarian activin:inhibin ratio.
Follicle stages
Secondary, Antral, Corpus luteum
Comment
Xu J, et al 2002 reported a stage-specific expression of smad2 and smad3 during
folliculogenesis.
They have demonstrated that two Smad family members that function as mediators for both
activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in
large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal
cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in
response to treatment with either TGFbeta or activin. However, Smad2 is more
responsive to activin stimulation, and Smad3 is more responsive to TGFbeta
stimulation. Stage-specific expression and differing ligand sensitivity of signaling
molecules may work together to allow different effects of TGFbeta family ligands
using the same signaling pathways over the course of follicular development.
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: None
type: null mutation fertility: embryonic lethal Comment:Waldrip et al. (1998) studied the effect of Smad2 in mouse embryonic development by targeted disruption of the mouse
Smad2 gene using embryonic stem cell technology. They found that Smad2 function was not required for mesoderm
production per se, but, rather unexpectedly, in the absence of Smad2, the entire epiblast adopts a mesodermal fate giving
rise to a normal yolk sac and fetal blood cells. In contrast, Smad2 mutant mouse embryos entirely lacked tissues of the
embryonic germ layers.
Species: mouse
Mutation name: None
type: null mutation fertility: subfertile Comment: Redundant Roles of SMAD2 and SMAD3 in Ovarian Granulosa Cells in vivo. Li Q et al. Transforming growth factor beta (TGFbeta) superfamily members are critical in maintaining cell growth and differentiation in the ovary. Although signaling of activins, TGFbetas, growth differentiation factor 9, and nodal converge preferentially to SMAD2 and SMAD3, the in vivo functions and redundancy of these SMADs in the ovary and female reproduction remain largely unidentified. To circumvent the deleterious phenotypic aspects of ubiquitous deletion of Smad2 and Smad3, a conditional knockout strategy was formulated to selectively inactivate Smad2, Smad3, or both Smad2 and Smad3 in ovarian granulosa cells. While granulosa cell ablation of individual Smad2 or Smad3 caused insignificant changes in female fertility, deletion of both Smad2 and Smad3 led to dramatically reduced female fertility and fecundity. These defects were associated with the disruption of multiple ovarian processes including follicular development, ovulation, and cumulus cell expansion. Furthermore, the impaired expansion of cumulus cells may be partially associated with altered cumulus expansion-related transcripts that are regulated by SMAD2/3 signaling. Our results indicate that SMAD2 and SMAD3 function redundantly in vivo to maintain normal female fertility and further support the involvement of an intraovarian SMAD2/3 pathway in mediating oocyte-produced signals essential for coordinating key events of the ovulatory process.