Activins are critical modulators of growth and survival

Activins betaA and betaB (encoded by Inhba and Inhbb genes, respectively) are related members of the TGF-beta superfamily. Previously, we generated mice with an Inhba knock-in allele (InhbaBK) that directs the expression of activin betaB protein in the spatiotemporal pattern of activin betaA. These mice were small and had shortened life spans, both influenced by the dose of the hypomorphic InhbaBK allele. To understand the mechanism(s) underlying these abnormalities, we now examine growth plates, liver, and kidney and analyze IGF-I, GH, and major urinary proteins. Our studies show that activins modulate the biological effects of IGF-I without substantial effects on GH, and that activin signaling deficiency also has modest effects on hepatic and renal function. To assess the relative influences of activin betaA and activin betaB, we produced mice that express activin betaB from the InhbaBK allele, and not from its endogenous Inhbb locus. InhbaBK/BK, Inhbb-/- mice have failure of eyelid fusion at birth and demonstrate more severe effects on somatic growth and survival than either of the corresponding single homozygous mutants, showing that somatic growth and life span are supported by both activins betaA and betaB, although activin betaA plays a more substantial role.     

Developmental profiles of activin betaA, betaB, and follistatin expression in the zebrafish ovary: evidence for their differential roles during sexual maturation and ovulatory cycle

Our recent experiments showed that gonadotropin(s) stimulated activin betaA and follistatin expression through the cAMP-PKA pathway but suppressed betaB via a cAMP-dependent but PKA-independent pathway in cultured zebrafish follicle cells. Given that pituitary gonadotropins are the major hormones controlling the development and function of the ovary, the differential expression of activin betaA and betaB as well as follistatin in response to gonadotropin(s) raises an interesting question about the temporal expression patterns of these molecules in vivo during sexual maturation and ovulatory cycle. Three experiments were performed in the present study. In the first experiment using sexually immature zebrafish, we followed the expression of activin betaA, betaB, and follistatin at the whole ovary level during a 10-day period in which the ovary developed from the primary growth stage to the one with nearly full-grown follicles. Activin betaA expression was very low at the primary growth stage but significantly increased with the growth of the ovary, and its rise was accompanied by an increase in follistatin expression. In contrast, the expression of activin betaB could be easily detected in the ovary of all stages; however, it did not exhibit an obvious trend of variation during the development. The second experiment examined the stage-dependent expression of activin betaA, betaB, and follistatin at the follicle level in the adult mature zebrafish. The expression of activin betaA was again low in the follicles during the primary growth stage, but exhibited a phenomenal increase after the follicles entered vitellogenesis with the peak level reached at midvitellogenic stage; in contrast, activin betaB mRNA could be easily detected at all stages with a slight increase during follicle growth. The expression of follistatin, on the other hand, also increased significantly during vitellogenesis; however, its level dropped sharply after reaching the peak at the midvitellogenic stage. In the third experiment, we investigated the dynamic changes of the ovarian activin betaA, betaB, and follistatin expression during the daily ovulatory cycle. The expression of activin betaA and follistatin gradually increased from 1800 h onward and reached the peak level around 0400 h when the germinal vesicles had migrated to the periphery in the full-grown oocytes. In contrast, activin betaB expression steadily declined, although not statistically significant, during the same period, but increased sharply at 0700 h when mature oocytes started to appear in most of the ovaries collected. In conclusion, activin betaA and betaB exhibit distinct expression patterns during the development of the ovary and the daily ovarian cycle of the zebrafish. It seems that activin betaA is involved in promoting ovary and follicle growth, whereas activin betaB may have a tonic role throughout follicle development but becomes critical at the late stage of oocyte maturation and/or ovulation.     

Interrelationship of growth differentiation factor 9 and inhibin in early folliculogenesis and ovarian tumorigenesis in mice

To investigate the interrelationship of inhibin alpha and growth differentiation factor 9 (GDF9) during early folliculogenesis, we generated mice lacking both inhibin alpha and GDF9. Our findings on these Inha Gdf9 double-mutant mice are as follows: 1). females develop ovarian tumors and a cachexia-like wasting syndrome, resembling mice lacking inhibin alpha alone. This indicates that the granulosa cells are competent to proliferate despite the lack of GDF9; 2). follicular development progresses to multiple-layer follicle stages before tumorigenesis. This demonstrates that the up-regulation of inhibin alpha in the Gdf9 knockout ovary directly prevents the proliferation of the granulosa cells at the primary follicle stage, an effect that is released in the absence of inhibin alpha; 3). a morphological theca forms around the preantral follicles with no detectable selective theca markers [i.e. 17alpha-hydroxylase (Cyp17), LH receptor (Lhr), and Kit]. These results indicate that the theca recruitment can occur independently of GDF9, but the differentiation of thecal cells is blocked; and 4). inhibin/activin subunits betaA, betaB, and Kit ligand (Kitl) mRNA are highly up-regulated, suggesting that the increased activins and KITL play functional roles in early folliculogenesis. Thus, GDF9 appears to function indirectly to regulate early granulosa cell proliferation and theca recruitment in vivo.     

Changes in the reproductive functions of mice due to injection of a plasmid expressing an inhibin alpha-subunit into muscle: a transient transgenic model

Inhibin is a gonadal hormone composed of an a-subunit and one of two beta-subunits (betaA, betaB), and its primary role is to inhibit FSH secretion by the pituitary. To investigate the roles of inhibin alpha in the reproductive system, an expression plasmid, pCMV-rINA, with the rat inhibin alpha cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Inhibin alpha mRNA was detected in the muscle by RT-PCR and Southern blot analysis. Inhibin protein was also detected, and Western blot analysis revealed a relatively high level of serum inhibin, but not of activin betaA. The estrous cycle of the pCMV-rINA-injected mice was extended, but there was no change in levels of pituitary FSH mRNA or serum FSH and no ovarian cysts were observed. When injected female mice were mated with males of proven fertility, litter size increased. Surprisingly, the embryos of pregnant females injected with pCMV-rINA, were retarded in growth and had defects in internal organs. When male mice were injected, testicle weight increased slightly without any noticeable change in the histology of the seminiferous tubules. Taken together, our data indicate that the inhibin alpha subunit influences a number of the reproductive functions of female mice.     

Control of digit formation by activin signalling

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.     

Cyclin D2 and p27 are tissue-specific regulators of tumorigenesis in inhibin alpha knockout mice

Inhibins are heterodimeric (alpha:betaA and alpha:betaB) endocrine, paracrine, and autocrine factors of the TGFbeta superfamily that are produced predominantly by ovarian granulosa cells in females and testicular Sertoli cells in males. Control of granulosa and Sertoli cell proliferation is lost in the inhibin alpha (Inhalpha) knockout mouse model, leading to gonadotropin-dependent gonadal tumors of the granulosa/Sertoli cell lineage in both females and males. Castrate Inhalpha knockout mice develop sex steroidogenic tumors of the adrenal cortex. Physiological control of granulosa/Sertoli cell cycle progression depends on p27Kip1 and cyclin D2, which function in the G1-->S phase transition. To study the cell cycle-regulatory factors involved in ovarian, testicular, and adrenal tumor development in vivo, we have bred Inhalpha mutant mice to mice with targeted disruptions of the p27 and cyclin D2 genes. Our previous studies demonstrated that inhibins act cooperatively with p27 to negatively regulate granulosa cell proliferation, as double mutant mice lacking inhibins and p27 develop and succumb to ovarian tumors more rapidly than Inhalpha knockout mice. Here, we report that cyclin D2 antagonizes this inhibition and is key in promoting gonadal growth and tumor development, and tumor development is markedly suppressed in double-mutant mice. We found that double-knockout females lacking cyclin D2 and Inhalpha lived longer than mice lacking inhibins alone; the majority of these double-knockout mice lived longer than 17 wk, as opposed to inhibin alpha single-knockout females with 50% survival at between 12 and 13 wk of age. Moreover, 95% of inhibin alpha knockout males succumb to testicular tumor development by 12 wk of age, whereas double knockouts were protected from early signs of tumor development and had a 50% survival of 40 wk. Interestingly, the results of these studies reflect tissue-specific consequences of loss of these cell cycle regulators. In castrate mice, loss of p27 has little effect on adrenal cortical tumor progression in the absence of inhibins, whereas loss of cyclin D2 prolongs the lifespan of cyclin D2, Inhalpha double knockouts. After gonadectomy, 50% of cyclin D2, Inhalpha double-knockout males live to more than 46 wk of age, 10 wk longer than 50% of littermates lacking only inhibins. Similarly, 50% of female cyclin D2, inhibin alpha double knockouts live to 47 wk of age before succumbing to adrenal tumor development, in contrast to the 50% survival of Inhalpha single-knockout females at between 27 and 28 wk. Thus, identification of genetic modifiers of the Inhalpha knockout tumor phenotype has led us to a better appreciation of how specific components of the cell cycle machinery contribute to tumorigenesis in the ovary, testis, and adrenal gland.     

Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

Background  

Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage.     

Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.     

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.     

Activin family members in the developing chick retina: expression patterns, protein distribution, and in vitro effects.

We have investigated whether the activin family of growth factors is involved in the regulation of retinal cell differentiation. Immunocytochemistry and in situ hybridization have shown that activin/inhibin subunits alpha, betaA, and betaB; receptors II and IIB; follistatin; and a follistatin-like gene are expressed in different regions of the chick embryo retina in developmentally regulated patterns. When tested in dissociated retinal cultures, activin did not appear to affect cell survival or proliferation, but it exerted marked inhibitory effects on the differentiation of photoreceptors, while stimulating the differentiation of nonphotoreceptor neurons; both effects were concentration-dependent and follistatin-sensitive. The results are consistent with the possibility that activin family members play significant roles in the regulation of retinal development.     

Spatial expression patterns of activin and its signaling system in the zebrafish ovarian follicle: evidence for paracrine action of activin on the oocytes

We have previously demonstrated that activin is likely an ovarian mediator of pituitary gonadotropin(s) and local epidermal growth factor in their stimulating oocyte maturation and maturational competence in the zebrafish. However, the downstream events controlled by activin remain unknown. One possible mechanism is that activin may directly work on the oocytes to promote the development of oocyte maturational competence. To substantiate this hypothesis, we performed the present study to demonstrate the expression of the activin system in different compartments of zebrafish follicles, namely, the follicle cells and oocytes. The proteins examined include activin subunits (betaA and betaB), activin-binding protein (follistatin), activin type II receptors (type IIA and IIB), the type I activin receptor-like kinases (ALK1-like, ALK2-like, and ALK4-like), and the intracellular activin signaling molecules (Smad2, Smad3, Smad4, and Smad7). The results showed that the entire activin signaling system is expressed by the full-grown immature zebrafish oocytes ( approximately 0.65 mm in diameter), including ALK4-like (ActRIB), ALK2-like (ActRIA), ActRIIA, ActRIIB, Smad2, Smad3, Smad4, and Smad7, therefore supporting our hypothesis that the oocytes are one of the direct targets of activin actions in the zebrafish ovary. In contrast, activin itself (betaA and betaB) and ALK1-like type I receptor are predominantly expressed in the follicle cells surrounding the oocytes. Interestingly, although follistatin is expressed in both the follicle cells and oocytes, its level of expression is significantly higher in the oocytes than the follicle cells, implying that follistatin may serve as a signal from the oocytes to modulate the activity of activin produced by the follicle cells. Taken together, the present study provides convincing evidence that although all members of the activin system are expressed in the whole follicle, they exhibit distinct spatial patterns of expression among different compartments of the follicle. It is likely that activin works directly on the oocytes in a paracrine manner to promote oocyte maturation and maturational competence. On the other hand, instead of being controlled passively by the follicle cells, the oocytes may actively participate in the regulation of follicle development by releasing various modulating molecules such as follistatin.     

Developmental changes in inhibin-alpha gene expression in the mouse testis

Inhibin is a gonadal hormone which is composed of an alpha-subunit and one of two related beta-subunits (betaA, betaB). Inhibin is important for pituitary FSH regulation, normal follicle development and maintenance of the estrous cycle in the female, whereas the role of inhibin in the male is less clear. Thus, we examined the expression of the inhibin-alpha gene in testis during sexual maturation in male mice, to try to gain insight into its functions in the male. Male mice of the ICR strain attained fertility at 6 weeks of age, and histological analysis revealed that a functional testis was formed, with seminiferous tubules which contain mature sperm and with an abundant population of Leydig cells. Parallel with this sexual maturation, inhibin-alpha subunit protein synthesis increased, whereas synthesis of the activin betaA and activin betaB followed with a delayed time course. Inhibin-alpha mRNA also increased during this critical period, and this corresponded to a change in the methylation status of the inhibin-alpha gene. Taken together, our data reveal that activation of inhibin-alpha gene during testis development correlated with the histological maturation of the testis and the acquisition of fertility in male mice.     

Should activin betaC be more than a fading snapshot in the activin/TGFbeta family album?

The activin growth factors consist of dimeric proteins made up of activin beta subunits and have been shown to be essential regulators of diverse systems in physiology. Four subunits are known to be expressed in mammalian cells: betaA, betaB, betaC, and betaE. Surprisingly, deletion of activin betaC and betaE subunits in vivo does not affect embryonic development or adult physiology which has led to the activin betaC and betaE subunits being regarded as non-essential and unimportant. The steady accumulation of circumstantial evidence to the contrary has led this lab to reassess the role of the activin betaC subunit. Activin betaC protein is expressed more widely than indicated by mRNA localisation. Experiments overexpressing activin betaC subunit or adding exogenous Activin C in vitro are contradictory but suggest roles for activin betaC in regulating Activin A action in apoptosis and homeostasis. Sequestration of betaA subunits by dimerisation with betaC subunits to form Activin AC represents an intracellular regulator of Activin A bioactivity. Activins play a pivotal role in normal physiology and carcinogenesis, so any molecule, such as the activin betaC subunit, that can affect activin action is potentially significant. Advancing our understanding of the physiological role of the activin betaC subunit requires new tools and reagents. Direct detection of the Activin AC dimer will be essential and will necessitate the purification of heteromeric Activin AC protein. In addition, there is a need for the development of an in vivo model of activin betaC subunit overexpression. With development of these tools, research into activin action in development and physiology can expand to include the less well understood members of the activin family such as activin betaC.     

Regulable expression of inhibin A in wild-type and inhibin alpha null mice

Exogenous regulation of protein expression creates the potential to examine the consequences of homeostatic Dysregulation in many physiological systems and, when used in transgenic mice, provides the capability of restoring a gene product to its knockout background without antigenicity issues. In this study, we used a mifeprisone-inducible system (the GeneSwitch system) to regulate the expression of inhibin A from the liver of mice. Inhibin is a heterodimeric protein (alpha/beta) wherein one of its subunits (beta) is capable of homodimerizing to form its physiological antagonist, activin (beta/beta). Inhibin is also expressed in two forms, A and B, as determined by the subtype of beta-subunit that dimerizes with the alpha-subunit (alpha/betaA or alpha/betaB). To utilize the GeneSwitch system, transgenic transactivator mice with liver-specific expression of a mifepristone-activated chimeric nuclear receptor (GLVP) were crossed with transgenic target mice containing a GVLP-responsive promoter upstream of polio-virus IRES (internal ribosome entry site)-linked sequences coding for the alpha- and beta-subunits of inhibin A. This intercross produced "bigenic" mice capable of regulable expression of inhibin A from the liver. Overexpression of inhibin A in wild-type mice produced a phenotype wherein males had decreased testis size and females had a block in folliculogenesis at the early antral stage, findings similar to activin type IIA receptor (ActRIIA) null mice. These phenotypes were most likely due to suppressed serum FSH, confirming that the liver-derived inhibin A was secreted into the serum to down-regulate pituitary FSH levels. Furthermore, the generation of bigenic mice in the inhibin alpha null background allowed for the induction of inhibin A in inhibin alpha null male mice with subsequent rescue of these mice from their gonadal tumor-induced lethal phenotype. This work demonstrates the in vivo production of a heterodimeric hormone from a single inducible promoter to study its therapeutic and physiological effects. In addition, these studies are the first example of an inducible system being used to prevent a lethal knockout phenotype in an animal model.     

The activin-follistatin system in the neonatal ovine uterus

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.     

Connective tissue growth factor is required for normal follicle development and ovulation

Connective tissue growth factor (CTGF) is a cysteine-rich protein the synthesis and secretion of which are hypothesized to be selectively regulated by activins and other members of the TGF-β superfamily. To investigate the in vivo roles of CTGF in female reproduction, we generated Ctgf ovarian and uterine conditional knockout (cKO) mice. Ctgf cKO mice exhibit severe subfertility and multiple reproductive defects including disrupted follicle development, decreased ovulation rates, increased numbers of corpus luteum, and smaller but functionally normal uterine horns. Steroidogenesis is disrupted in the Ctgf cKO mice, leading to increased levels of serum progesterone. We show that disrupted follicle development is accompanied by a significant increase in granulosa cell apoptosis. Moreover, despite normal cumulus expansion, Ctgf cKO mice exhibit a significant decrease in oocytes ovulated, likely due to impaired ovulatory process. During analyses of mRNA expression, we discovered that Ctgf cKO granulosa cells show gene expression changes similar to our previously reported granulosa cell-specific knockouts of activin and Smad4, the common TGF-β family intracellular signaling protein. We also discovered a significant down-regulation of Adamts1, a progesterone-regulated gene that is critical for the remodeling of extracellular matrix surrounding granulosa cells of preovulatory follicles. These findings demonstrate that CTGF is a downstream mediator in TGF-β and progesterone signaling cascades and is necessary for normal follicle development and ovulation.     

Activin A stimulates type IV collagenase (matrix metalloproteinase-2) production in mouse peritoneal macrophages

The role of activin, a dimer of inhibin beta subunit, in mouse peritoneal macrophages was evaluated. Activin activity in the cultured macrophages was augmented in response to activation by LPS. In Western blot analysis, immunoreactive activin A was detected in the culture medium only when the macrophages were stimulated by LPS. Although mRNA expression of betaA subunit was detected, that of alpha and betaB subunit was not found in macrophages by reverse RT-PCR. The activin betaA mRNA level was increased in macrophages by LPS, suggesting that the activin production augmented by LPS is regulated at the mRNA level of the betaA gene. The mRNAs of four activin receptors (ActRI, ActRIB, ActRII, and ActRIIB) were also detected in the peritoneal macrophages, and the mRNA levels, except for ActRIB, were decreased during the LPS treatment. Exogenous activin A stimulated the mRNA expression and gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) in macrophages in both the presence and the absence of LPS. In contrast, activin did not affect the production of MMP-9 in macrophages. These results suggested that 1) mouse peritoneal macrophages produced activin A; 2) expression of activin A was enhanced with activation of the macrophages; 3) the macrophages also expressed activin receptors; and 4) exogenous activin A stimulated MMP-2 expression and activity, implicating activin A as an positive regulator of MMP-2 expression. Considering that MMP-2 constitutes the rate-limiting proteinase governing the degradation of basement membrane collagens, activin A may be involved in migration and infiltration of macrophages through the basement membrane in an inflammatory state.     

The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development.

The gene for activin betaA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, we have explored the role of mediators of activin signalling in tooth development. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin betaA mutants can be observed. In addition, we identify a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, and show that its expression in activin betaA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other transforming growth factor beta molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development, which reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars.