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.

Characterisation of the porcine cytokines which activate the CD131βc common sub-unit, for potential immune-augmentation

Early acting cytokines and growth factors such as those of the CD131 βc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

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.

CBL Linker Region and RING Finger Mutations Lead to Enhanced Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Signaling via Elevated Levels of JAK2 and LYN

Juvenile myelomonocytic leukemia (JMML) is characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). SHP2, NF-1, KRAS, and NRAS are mutated in JMML patients, leading to aberrant regulation of RAS signaling. A subset of JMML patients harbor CBL mutations associated with 11q acquired uniparental disomy. Many of these mutations are in the linker region and the RING finger of CBL, leading to a loss of E3 ligase activity. We investigated the mechanism by which CBL-Y371H, a linker region mutant, and CBL-C384R, a RING finger mutant, lead to enhanced GM-CSF signaling. Expression of CBL mutants in the TF-1 cell line resulted in enhanced survival in the absence of GM-CSF. Cells expressing CBL mutations displayed increased phosphorylation of GM-CSF receptor βc subunit in response to stimulation, although expression of total GM-CSFR βc was lower. This suggested enhanced kinase activity downstream of GM-CSFR. JAK2 and LYN kinase expression is elevated in CBL-Y371H and CBL-C384R mutant cells, resulting in enhanced phosphorylation of CBL and S6 in response to GM-CSF stimulation. Incubation with the JAK2 inhibitor, TG101348, abolished the increased phosphorylation of GM-CSFR βc in cells expressing CBL mutants, whereas treatment with the SRC kinase inhibitor dasatinib resulted in equalization of GM-CSFR βc phosphorylation signal between wild type CBL and CBL mutant samples. Dasatinib treatment inhibited the elevated phosphorylation of CBL-Y371H and CBL-C384R mutants. Our study indicates that CBL linker and RING finger mutants lead to enhanced GM-CSF signaling due to elevated kinase expression, which can be blocked using small molecule inhibitors targeting specific downstream pathways.

Abstract 1070: Differential response dynamic of leukemia cells to GM-CSF and IL-3 stimulation in signal transduction pathways

Abstract GM-CSF and IL-3 regulate the survival, proliferation, differentiation, and functional activation of hematopoietic cells. GM-CSF also contributes to controlling the function of dendritic cells and T-cells. Aberrant function of GM-CSF has been linked to multiple diseases including juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia, rheumatoid arthritis, and alveolar proteinosis. GM-CSF and IL-3 share a common βc subunit on their receptor. Many studies have revealed that dys-regulation of Ras/PI3K/Akt and Ras/Raf/Mek/Erk pathways are responsible for hypersensitivities of GM-CSF and IL-3 in various diseases. However, few data have distinguished the difference of signal transduction pathways between the two cytokines. We previously reported that JMML cells are selectively hypersensitive to GM-CSF in vitro, while they show normal sensitivity to IL-3. Others documented that there was a time gap between CREB responded to GM-CSF and IL-3 stimulation. In the present study, we hypothesized that the signal transduction pathway of GM-CSF has differential response dynamic from that of IL-3. We first tested the CFU-GM growth pattern of a leukemia cell line, TF-1, in response to GM-CSF and IL-3 stimulation. We found that the GM-CSF dose-response curve was markedly shifted leftward, indicating that TF-1 cells were more sensitive to GM-CSF than to IL-3. This pattern is very similar to that observed in primary JMML cells. We next investigated the activities of the elements downstream in Ras pathway. After serum starving in medium with 0.5% BSA for 16 hours, TF-1 cells were stimulated with GM-CSF or IL-3 at a concentration range of 0.01-500pM. We found that the total protein levels of CREB were consistently unchanged. On the other hand, significant phosphorylation of CREB on serine 133 began to occur at concentrations of GM-CSF at 10pM. Significant phosphorylation of Erk was observed even at a concentration as low as 1 pM of GM-CSF. However, this phosphorylation pattern was not seen until 100pM of IL-3, and the level of phosphorylation was lower. The same dynamic, GM-CSF vs. IL-3, was also found in pAkt and pSTAT5. In conclusion, our data demonstrate that leukemia cells respond to GM-CSF and IL-3 stimulation in different dynamic patterns in signal transduction pathways. This indicates that the selective hypersensitivity to GM-CSF in some leukemia cells may be caused by aberrant elements in signal transduction pathways that are responsive to the low concentrations of GM-CSF stimulation. We raise a concern that any study related to responsiveness of GM-CSF or IL-3 stimulation should be interpreted cautiously; with specific attention paid to the concentration levels and the length of stimulation, in order to precisely characterize the function of GM-CSF and IL-3. This observed differential response dynamic might apply to a broader phenomenon in other cytokines or hormones in other tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1070. doi:1538-7445.AM2012-1070

Molecular and clinical rationale for therapeutic targeting of interleukin‐5 and its receptor

Interleukin-5 is a Th2 homodimeric cytokine involved in the differentiation, maturation, migration, development, survival, trafficking and effector function of blood and local tissue eosinophils, in addition to basophils and mast cells. The IL-5 receptor (IL-5R) consists of an IL-5-specific α subunit that interacts in conformationally dynamic ways with the receptor's βc subunit, an aggregate of domains it shares with binding sites of IL-3 and granulocyte-macrophage colony-stimulating factor. IL-5 and IL-5R drive allergic and inflammatory immune responses characterizing numerous diseases, such as asthma, atopic dermatitis, chronic obstructive pulmonary disease, eosinophilic gastrointestinal diseases, hyper-eosinophilic syndrome, Churg-Strauss syndrome and eosinophilic nasal polyposis. Although corticosteroid therapy is the primary treatment for these diseases, a substantial number of patients exhibit incomplete responses and suffer side-effects. Two monoclonal antibodies have been designed to neutralize IL-5 (mepolizumab and reslizumab). Both antibodies have demonstrated the ability to reduce blood and tissue eosinophil counts. One additional monoclonal antibody, benralizumab (MEDI-563), has been developed to target IL-5R and attenuate eosinophilia through antibody-dependent cellular cytotoxicity. All three monoclonal antibodies are being clinically evaluated. Antisense oligonucleotide technology targeting the common βc IL-5R subunit is also being used therapeutically to inhibit IL-5-mediated effects (TPI ASM8). Small interfering RNA technology has also been used therapeutically to inhibit the expression of IL-5 in animal models. This review summarizes the structural interactions between IL-5 and IL-5R and the functional consequences of such interactions, and describes the pre-clinical and clinical evidence supporting IL-5R as a therapeutic target.

Immunohistochemical Labeling of the Inhibin/Activin βC Subunit in Normal Human Placental Tissue and Chorionic Carcinoma Cell Lines

Inhibins and activins are important regulators of the female reproductive system. A novel inhibin subunit, named betaC, has been identified and demonstrated to be expressed in several human tissues. We demonstrate here that inhibin betaC is expressed in human placenta. Expression of the inhibin betaC subunit was demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by an inhibin betaC-specific RT-PCR analysis. Expression of inhibin betaC was detected in the human chorionic carcinoma cell lines JEG and BeWo. Although the precise role of this novel inhibin subunit in human placenta development and homeostasis is unclear, analogies with other inhibin subunits and the strong expression of betaC in normal human trophoblast cells and chorionic carcinoma cells suggest that betaC may be involved in autocrine/paracrine signaling pathways, angiogenesis, decidualization, and tissue remodeling under normal and malignant conditions. Additionally, JEG and BeWo express betaC and, therefore, can be used as a cell culture model for further functional analysis of this subunit in the human placenta.

Genetic engineering of pharmacologically regulated T cells, specific for breast cancer target antigens

Peripheral blood T cells can be genetically targeted against cancer using fusion receptors known as chimeric antigen receptors (CARs). Many preclinical studies have provided great encouragement for this approach. However, pioneering clinical trials have been less successful and identified poor T-cell survival in patients as a crucial limiting factor. To address this, what is needed is a system to achieve selective expansion of tumour-specific effector T cells, both in vitro and in vivo. Here, we describe such an approach using IL-4, a pharmaceutical that has been tested in cancer patients and which is normally a poor mitogen for T cells. A chimeric cytokine receptor named 4αβ was engineered in which the IL-4 receptor α (IL-4Rα) ectodomain was fused to the shared βc subunit, used by IL-2/IL-15. Addition of IL-4 to 4αβ-expressing T cells resulted in selective phosphorylation of STAT3/STAT5/ERK, mimicking the actions of IL-2 or IL-15. Using receptor-selective IL-4 muteins, partnering of 4αβ with γc was implicated in these findings. Next, human T cells were engineered to co-express 4αβ with CARs specific for two breast cancer targets: MUC1 or the extended ErbB family. These T cells exhibited an unprecedented capacity to undergo IL-4-dependent expansion in vitro and repeatedly destroyed breast cancer cultures, greatly exceeding the performance of IL-2-stimulated cells. Importantly, 4αβ-expressing T cells retained cytolytic specificity for target antigen and dependence upon IL-4 (or IL-2) for survival. We have also used this system to achieve rapid IL-4-driven ex vivo expansion and enrichment of CAR+ human T cells in bags (T-bags). Experiments were performed under closed and pseudo-good manufacturing practice conditions, scaling up for phase 1 clinical trials. T cells expanded in this manner demonstrate Th1 polarisation and potent tumour destructive activity, both in vitro and in vivo, in tumour-bearing SCID Beige mice. Together, these findings provide proof of principle for the development of pharmacologically regulated T-cell immunotherapy for breast and other cancers.

Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML

AbstractDeregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34+CD38−CD123+ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

The IL‐3/IL‐5/GM‐CSF Common β Receptor Plays a Pivotal Role in Regulating Th2 Immunity and Allergic Airway Inflammation

The eosinophil is a central effector cell in allergic asthma. Differentiation and function of eosinophils is regulated by interleukin (IL)‐3, IL‐5 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), which all signal through a common β receptor subunit (βc). Recent therapeutic approaches targeting IL‐5 alone have had limited effects on disease progression. By employing mice lacking the βc and βIL‐3 receptor subunits we were able to interrupt signalling through all three eosinophilopoietic cytokines and prevent allergen‐induced expansion and accumulation of eosinophils in the lung in a mouse model of allergic airways inflammation. Moreover, βc deficiency resulted in inhibition of hallmark features of asthma including airways hypersensitivity, mucus hypersecretion and antigen‐specific IgE. Surprisingly, we also identified a critical role for the βc in regulating type 2 immunity. Th2 cells in the lung of allergen‐challenged βc−/− mice were limited in their ability to proliferate, produce cytokines and migrate to effector sites, which was attributed to reduced numbers of myeloid dendritic cells in the lung compartment. Thus the βc subunit plays a critical role in allergen‐induced eosinophil responses and is pivotal in regulating molecules that promote both early and late phases of allergic inflammation, representing a novel target for therapy. (Supported by the NIH and NHMRC)

The Shc-binding site of the βc subunit of the GM-CSF/IL-3/IL-5 receptors is a negative regulator of hematopoiesis

AbstractTyrosine and serine phosphorylation of the common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is widely viewed as a general mechanism that provides positive inputs by coupling the receptor to signaling pathways that stimulate several cellular functions. We show here that despite the known action of Tyr577 in βc to recruit Shc–PI-3 kinase (PI3K) pathway members, Tyr577 plays, surprisingly, a negative regulatory role in cell function, and that this is mediated, at least in part, through the uncoupling of SH2-containing inositol 5′-phosphatase (SHIP) from βc. Fetal liver cells from βc/βIL-3−/− mice expressing human GM-CSF receptor α chain and βc Tyr577Phe mutant showed enhanced colony formation and expansion of progenitor cells in response to GM-CSF. Dissection of these activities revealed that basal survival was increased, as well as cytokine-stimulated proliferation. As expected, the recruitment and activation of Shc was abolished, but interestingly, Gab-2 and Akt phosphorylation increased. Significantly, the activation of PI3K was enhanced and prolonged, accompanied by loss of SHIP activity. These results reveal a previously unrecognized negative signaling role for Tyr577 in βc and demonstrate that uncoupling Shc from cytokine receptors enhances PI3K signaling as well as survival and proliferation.

Reactive oxygen species generated by hematopoietic cytokines play roles in activation of receptor-mediated signaling and in cell cycle progression

Hematopoietic cytokines, including interleukin (IL)-3 and erythropoietin (Epo), regulate hematopoiesis by stimulating their receptors coupled with the Jak2 tyrosine kinase to induce receptor tyrosine phosphorylation and activate mainly the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways. Here we demonstrate that IL-3 or Epo induces a rapid and transient (peaking at 30 min) as well as late progressive increase in reactive oxygen species (ROS) in a hematopoietic progenitor model cell line, 32Dcl3, and its subclone expressing the Epo receptor (EpoR), 32D/EpoR-Wt. The cytokine-induced ROS generation was not affected in 32Dcl3 cells depleted of mitochondrial DNA. The antioxidant N-acetyl- l-cysteine (NAC) inhibited IL-3-induced tyrosine phosphorylation of Jak2, IL-3 receptor βc subunit (IL-3Rβc), and STAT5 as well as activation-specific phosphorylation of Akt, MEK, and ERK, while treatment of cells with H 2O 2 activated these signaling events. NAC also inhibited the EpoR-induced transphosphorylation of IL-3Rβc. Moreover, NAC treatment reduced the expression levels of c-Myc, Cyclin D2, and Cyclin E, and induced expression of p27, thus inhibiting the G1 to S phase transition of cells cultured with IL-3. Further studies have shown that the degradation of c-Myc was facilitated or inhibited by treatment of cells with NAC or H 2O 2, respectively. These data indicate that the rapid generation of ROS by cytokine stimulation, which is at least partly independent of mitochondria, may play a role in activation of Jak2 and the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways as well as in transactivation of cytokine receptors. The cytokine-induced ROS generation was also implicated in G1 to S progression, possibly through stabilization of c-Myc and induction of G1 phase Cyclin expression leading to suppression of p27.

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.

Alternative strategies for antagonism of IL-5

RATIONALE: Interleukin-5 (IL-5) is crucial for the differentiation and bioactivity of eosinophils. Signaling is mediated by IL-5 ligation of its heterodimeric receptor comprised of the cytokine-specific IL-5Rα subunit and the βc subunit. The hypothesis that alternative therapeutic strategies for antagonism of IL-5 would have distinct inhibitory properties was investigated.

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.

Integrins on eggs: the βC subunit is essential for formation of the cortical actin cytoskeleton in sea urchin eggs

Eggs of several metazoans have been demonstrated to express integrins; however, their function is unclear. Previous studies have shown that the βC integrin subunit is expressed on unfertilized sea urchin eggs and proteolytically removed at fertilization. Here we report that the βC subunit is reexpressed on the egg surface immediately after fertilization. Using morpholino antisense oligonucleotides to block translation, we show that without βC expression, eggs undergo cleavage resulting in loosely adherent cells that fail to develop beyond a blastula. Without βC containing integrins, the cortical actin network of the egg does not form, yet contractile rings appear. Coinjection of RNA encoding the βC or chicken β1 subunit, but lacking the morpholino target sequence, rescues the cortical actin network and normal embryos result. Coinjection of RNA encoding the βC subunit lacking the cytoplasmic domain fails to rescue. These studies demonstrate that the cortical actin cytoskeleton is anchored by βC integrins and contractile ring actin is not. We suggest that one important function of egg integrins is to organize the actin cortex.

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.

Structure of the Complete Extracellular Domain of the Common β Subunit of the Human GM-CSF, IL-3, and IL-5 Receptors Reveals a Novel Dimer Configuration

The receptor systems for the hemopoietic cytokines GM-CSF, IL-3, and IL-5 consist of ligand-specific α receptor subunits that play an essential role in the activation of the shared βc subunit, the major signaling entity. Here, we report the structure of the complete βc extracellular domain. It has a structure unlike any class I cytokine receptor described thus far, forming a stable interlocking dimer in the absence of ligand in which the G strand of domain 1 hydrogen bonds into the corresponding β sheet of domain 3 of the dimer-related molecule. The G strand of domain 3 similarly partners with the dimer-related domain 1. The structure provides new insights into receptor activation by the respective α receptor:ligand complexes.