Activin βC Subunit Research Articles

Transcriptional Profiling of TGF-β Superfamily Members in Lumbar DRGs of Rats Following Sciatic Nerve Axotomy and Activin C Inhibits Neuropathic Pain.

Neuroinflammation and cytokines play critical roles in neuropathic pain and axon degeneration/regeneration. Cytokines of transforming growth factor-β superfamily have implications in pain and injured nerve repair processing. However, the transcriptional profiles of the transforming growth factor-β superfamily members in dorsal root ganglia under neuropathic pain and axon degeneration/regeneration conditions remain elusive. We aimed to plot the transcriptional profiles of transforming growth factor-β superfamily components in lumbar dorsal root ganglia of sciatic nerve-axotomized rats and to further verify the profiles by testing the analgesic effect of activin C, a representative cytokine, on neuropathic pain. Adult male rats were axotomized in sciatic nerves, and lumbar dorsal root ganglia were isolated for total RNA extraction or section. A custom microarray was developed and employed to plot the gene expression profiles of transforming growth factor-β superfamily components. Realtime RT-PCR was used to confirm changes in the expression of activin/inhibin family genes, and then in situ hybridization was performed to determine the cellular locations of inhibin α, activin βC, BMP-5 and GDF-9 mRNAs. The rat spared nerve injury model was performed, and a pain test was employed to determine the effect of activin C on neuropathic pain. The expression of transforming growth factor-β superfamily cytokines and their signaling, including some receptors and signaling adaptors, were robustly upregulated. Activin βC subunit mRNAs were expressed in the small-diameter dorsal root ganglion neurons and upregulated after axotomy. Single intrathecal injection of activin C inhibited neuropathic pain in spared nerve injury model. This is the first report to investigate the transcriptional profiles of members of transforming growth factor-β superfamily in axotomized dorsal root ganglia. The distinct cytokine profiles observed here might provide clues toward further study of the role of transforming growth factor-β superfamily in the pathogenesis of neuropathic pain and axon degeneration/regeneration after peripheral nerve injury.

Cytokine activin C ameliorates chronic neuropathic pain in peripheral nerve injury rodents by modulating the TRPV1 channel.

The cytokine activin C is mainly expressed in small-diameter dorsal root ganglion (DRG) neurons and suppresses inflammatory pain. However, the effects of activin C in neuropathic pain remain elusive. Male rats and wild-type and TRPV1 knockout mice with peripheral nerve injury - sciatic nerve axotomy and spinal nerve ligation in rats; chronic constriction injury (CCI) in mice - provided models of chronic neuropathic pain. Ipsilateral lumbar (L)4-5 DRGs were assayed for activin C expression. Chronic neuropathic pain animals were treated with intrathecal or locally pre-administered activin C or the vehicle. Nociceptive behaviours and pain-related markers in L4-5 DRGs and spinal cord were evaluated. TRPV1 channel modulation by activin C was measured. Following peripheral nerve injury, expression of activin βC subunit mRNA and activin C protein was markedly up-regulated in L4-5 DRGs of animals with axotomy, SNL or CCI. [Correction added on 26 November 2020, after first online publication: The preceding sentence has been corrected in this current version.] Intrathecal activin C dose-dependently inhibited neuropathic pain in spinal nerve ligated rats. Local pre-administration of activin C decreased neuropathic pain, macrophage infiltration into ipsilateral L4-5 DRGs and microglial reaction in L4-5 spinal cords of mice with CCI. In rat DRG neurons, activin C enhanced capsaicin-induced TRPV1 currents. Pre-treatment with activin C reduced capsaicin-evoked acute hyperalgesia and normalized capsaicin-evoked persistent hypothermia in mice. Finally, the analgesic effect of activin C was abolished in TRPV1 knockout mice with CCI. Activin C inhibits neuropathic pain by modulating TRPV1 channels, revealing potential analgesic applications in chronic neuropathic pain therapy.

Role of activin C in normal ovaries and granulosa cell tumours of mice and humans.

Activins and inhibins play important roles in the development, growth and function of the ovary. Mice lacking inhibin develop granulosa cell tumours in their ovaries that secrete activin A, and these tumours are modulated by increased activin C expression. The aim of the present study was to identify where activin C is expressed in mouse and human ovaries and whether overexpression of activin C modulates normal follicular development in mice. Immunohistochemical staining for the activin βC subunit was performed on sections from mouse and human ovaries and human adult granulosa cell tumours. Stereology techniques were used to quantify oocyte and follicular diameters, and the percentage of different follicular types in ovaries from wild-type mice and those underexpressing inhibin α and/or overexpressing activin C. Staining for activin βC was observed in the oocytes, granulosa cells, thecal cells and surface epithelium of mouse and human ovaries, and in the granulosa-like cells of adult granulosa cell tumours. Overexpression of activin C in mice did not alter follicular development compared with wild-type mice, but it did modulate the development of abnormal early stage follicles in inhibin α-null mice. These results provide further evidence of a role for activin C in the ovary.

Over-Expression of Activin-βC Is Associated with Murine and Human Prostate Disease.

Activins are members of the TGF-β superfamily and have been linked to prostate cancer. There are four mammalian activin subunits (βA, βB, βC, and βE) that dimerize to form functional proteins. The role of activin-A (βA-βA) has been relatively well characterized and has been shown to generally inhibit growth in the prostate. In contrast, little is known about the biological function of the βC and βE subunits. Previous work indicated activin-C (βC-βC) to be an antagonist of activin-A. This is important because resistance to activin-A growth inhibition occurs during prostate cancer progression. This paradox is not currently well understood. Hence, we hypothesize that local expression of the activin-βC subunit antagonizes activin-A-dependent growth inhibition and represents a key factor contributing to acquired insensitivity to activin-A observed in prostate cancer progression. To test our hypothesis, we characterized the ventral prostate lobes of 9-month-old transgenic mice over-expressing activin-βC and examined the expression of activin-βA, activin-βC, and the activin intracellular signaling factor, Smad-2, in human prostate diseases. Prostate epithelial cell hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN) lesions, alterations in cell proliferation, and reduced Smad-2 nuclear localization were evident in mice over-expressing activin-βC. Increased activin-βA and -βC subunit immunoreactive scores and decreased Smad-2 nuclear localization were also evident in human prostate cancer. This study suggests that over-expression of activin-βC is associated with murine and human prostate pathologies. We conclude that the activin-βC subunit may have therapeutic and/or diagnostic implications in human prostate disease.

Regional localization of activin-βA, activin-βC, follistatin, proliferation, and apoptosis in adult and developing mouse prostate ducts

Activins and inhibins, members of the TGF-β superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-βA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-β superfamily, the activin-βC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (βC-βC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.

Activin‐βc reduces reproductive tumour progression and abolishes cancer‐associated cachexia in inhibin‐deficient mice

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α-subunit and by an activin-binding protein, follistatin. The activin-β(C) subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin-β(C) forms heterodimers with activin-β(A) and activin-C antagonizes activin-A in vitro. Thus, it is proposed that overexpression, rather than loss of activin-β(C) , regulates activin-A bioactivity. In order to prove biological efficacy, inhibin α-subunit knockout mice (α-KO) were crossed with mice overexpressing activin-β(C) (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin-A, and cancer-associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α-KO/ActC++ mice, where excessive activin-A is the underlying cause. Accordingly, a reduction in activin-A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α-KO/ActC++ mice. Overexpression of activin-β(C) antagonized the activin signalling cascade; thus, the tumourigenic effects of activin-A were abrogated. This study provides proof of the biological relevance of activin-β(C) . Being a regulator of activin-A, it is able to abolish cachexia and modulate reproductive tumour development in α-KO mice.

179. CHARACTERISATION OF THE IN VITRO FUNCTION OF ACTIVIN AC

Activins are members of the TGF-β superfamily that signal via type II and type I receptor subunits and intracellular Smads1. Activin A stimulates FSH release from the pituitary and is also a potent growth and differentiation factor in many physiological systems2. Over-expression of the activin-βC subunit in vitro leads to a reduction in activin A and an increase in activin AC3. Transgenic mice over-expressing activin-βC show decreased circulating activin A, implying that activin AC may also be formed in vivo4. Recently recombinant activin AC has become available, therefore this study examines the in vitro function and mechanism of action of activin AC. Activin AC stimulates FSH release in LβT2 cells and is a negative growth regulator in LNCaP cells, however the potency of activin AC is 8-10 fold less than activin A. Incubation of LNCaP cells with activin receptor antibodies (ALK4, ActRIIA, ActRIIB) abolishes the growth inhibitory effects of activin AC. Activin AC binds to ActRIIB, however a 20-30 fold decrease in both the potency and affinity of activin AC is evident compared to activin A. In addition, activin AC increases Smad-2 phosphorylation. These results indicate activin AC utilises the same receptors and intracellular signalling molecules as activin A. The activin A antagonists, follistatin and activin C4, also antagonise the growth inhibitory effects of activin AC and reduce Smad-2 phosphorylation and Smad-4 expression. This study shows for the first time that the in vitro function of activin AC is similar to activin A, albeit at a lower potency and provides the impetus to determine the physiological role of activin AC in vivo.

235. Activin C antagonises activin A in vitro and over-expression leads to prostate pathologies in vivo

Activin A is a well characterised inhibitor of proliferation in most epithelial cells. The actions of activin A on cell growth are mediated through Smad-dependent pathways. Activin A is potent at low levels, therefore its synthesis and bioactivity must be tightly regulated. Follistatin binding or inhibin subunit heterodimerisation block access to the activin receptor and/or receptor activation. We postulate that another mechanism of regulating activin A bioactivity is through the activin-βC subunit. In order to test our hypothesis produced recombinant activin C and mice overexpressing activin-βC. Recombinant activin C abrogated activin A-induced growth inhibition in vitro and the mechanism of action was downregulation of activin A-induced Smad signalling molecules. In the prostate overexpression of activin-βC increased epithelial cell proliferation while there was no significant difference in apoptotic epithelial cells. This imbalance between proliferation and apoptosis led to a significant increase in ventral prostate weight, prostatic hypertrophy and epithelial cell hyperplasia. A significant decrease in nuclear localisation of Smad-2 was associated with activin-βC overexpression in the prostate which implies antagonism of activin signalling also occurs in vivo. This is the first study to provide evidence that activin-βC is an antagonist of activin A in vitro and in vivo and implicates a role for the activin-βC subunit in maintenance of tissue homeostasis in the prostate.

Should activin βC be more than a fading snapshot in the activin/TGFβ family album?

The activin growth factors consist of dimeric proteins made up of activin β subunits and have been shown to be essential regulators of diverse systems in physiology. Four subunits are known to be expressed in mammalian cells: βA, βB, βC, and βE. Surprisingly, deletion of activin βC and βE subunits in vivo does not affect embryonic development or adult physiology which has led to the activin βC and βE 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 βC subunit. Activin βC protein is expressed more widely than indicated by mRNA localisation. Experiments overexpressing activin βC subunit or adding exogenous Activin C in vitro are contradictory but suggest roles for activin βC in regulating Activin A action in apoptosis and homeostasis. Sequestration of βA subunits by dimerisation with βC 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 βC subunit, that can affect activin action is potentially significant. Advancing our understanding of the physiological role of the activin βC 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 βC 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 βC.

119. Over-expression of activin βC in vivo reveals a role in male fertility

Introduction: Activin βC subunit antagonises the formation and bioactivity of activin A via intracellular heterodimerisation and decreases activation of the activin signalling pathway (Mellor et al. 2003). Therefore the activin βC subunit heterodimers provide a new mechanism of regulating activin levels. Vedja and colleagues over-expressed the activin βC subunit in malignant liver cell lines, which subsequently displayed inhibition of cell proliferation and induction of apoptosis (Vedja et al. 2003). Conversely, Wada et al. demonstrated that treatment with hr-activin C stimulates growth of a liver cell line (Wada et al. 2004). These recent (and contradictory) reports about the in vitro activity of activin βC have prompted us to examine the in vivo role of activin βC by creating a transgenic mouse over-expressing the βC activin subunit. Methods: The full-length human cDNA under the control of a CMV promoter was incorporated into the genome of three founder C57/B6 mice. Genotyping was performed by both Southern and PCR. Mice were monitored weekly and culled at 14–16 weeks (adult). Blood was collected by cardiac puncture, organs were weighed and a portion fixed in Bouin’s or frozen for subsequent RNA and protein extraction. Daily sperm production (DSP) was determined by standard methods. Sertoli and germ cell number will be determined using the optical disector (sic) stereological technique in Bouin’s fixed resin sections. Proliferation and apoptosis will be examined using PCNA and TUNEL respectively. Activin A was assessed by ELISA, while FSH, LH, follistatin and total inhibin were determined by RIA. Results and conclusions: Over-expression of activin-βC resulted in decreased circulating activin A (P < 0.005 TG1, P < 0.05 TG2 and P = 0.08 TG3), a progressive age-related decrease in litter sizes (9.3 WT v. 6.3 TG1, 5.8 TG2 and 4.5 TG3; P < 0.005 v. WT) and testicular DSP (P < 0.05). These data support the hypothesis that βC is a novel in vivo regulator and is the first indication of a role for activin-βC in male fertility. This novel mouse model will significantly advance our understanding of the in vivo role of activin-βC. (1)Mellor et al. (2003). Endocrinology 144, 4410–4419.(2)Vejda et al. (2003). Carcinogenesis 24, 1801–1809.(3)Wada et al. (2004). Am. J. Physiol. Endocrinol. Metab. 287, E247–E254.

Genes Highly Expressed in the Early Phase of Murine Graft-versus-Host Reaction

Graft-versus-host reaction (GVHR) is a complex process initiated upon allorecognition. For detection of early molecular events in GVHR, we first assessed time courses with respect to symptoms and serum interferon (IFN)-γ levels and then used the differential display method to compare gene transcript patterns during the early phase between acute lethal GVHR mice and syngeneic controls. In the GVHR mice, high expression levels of seven genes encoding the following molecules were detected: TGTP/Mg21 (an IFN-γ-related signaling molecule), vitronectin, Nedd5 (a mammalian septin), manganese superoxide dismutase, activin βC subunit, PRCC (a papillary renal cell carcinoma-associated molecule), and an uncharacterized gene corresponding to a mouse expressed sequence tag (EST). The expression levels of most genes peaked before the symptomatological onset and the peak of IFN-γ levels. Thus, gene expression monitoring may characterize the inductive process of GVHR and aid in the development of gene-based diagnostics and therapies.

Expression of activin beta C subunit mRNA in reproductive tissues.

A cDNA encoding the potential activin beta C subunit was produced from human testis RNA using reverse transcription and PCR and then used as a probe in Northern blot analysis of poly(A)+ RNA. A transcript of approximately 1.8 kb was evident in human ovary, placenta and testis samples. A transcript of this size was also detected in adult rat caput and cauda epididymis, and adult and day-15 testis and adult spleen, while adult liver contained a single transcript of approximately 2.1 kb. Poly(A)+ RNA from primary spermatocytes but not from round spermatids contained the 1.8 kb activin beta C mRNA. These findings highlight the need for further studies to determine the physiological role(s) of activin beta C in a wide variety of tissues.

Expression of activin receptor and follistatin, an activin-binding protein, in osteoblastic cells

Activins and inhibins, members of the TGF-β superfamily, are growth and differentiation factors involved in the regulation of several biological processes, including reproduction, development, and fertility. Previous studies have shown that the activin-βA subunit plays a pivotal role in prostate development. Activin-A inhibits branching morphogenesis in the developing prostate, and its expression is associated with increased apoptosis in the adult prostate. Follistatin, a structurally unrelated protein to activins, is an antagonist of activin-A. A balance between endogenous activin-A and follistatin is required to maintain prostatic branching morphogenesis. Deregulation of this balance leads to branching inhibition or excessive branching and increased maturation of the stroma surrounding the differentiating epithelial ducts. Recent work identified another member of the TGF-β superfamily, the activin-βC subunit, as a novel antagonist of activin-A. Over-expression of activin-C (βC-βC) alters prostate homeostasis, by interfering with the activin-A signaling. The current study characterized the spatiotemporal localization of activin-A, activin-C and follistatin in the adult and developing mouse prostate using immunohistochemical analysis. Results showed activin-C and follistatin are differentially expressed during prostate development and suggested that the antagonistic property of follistatin is secondary to the action of activin-C. In conclusion, the present study provides evidence to support a role of activin-C in prostate development and provides new insights in the spatiotemporal localization of activins and their antagonists during mouse prostate development.