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Activins and Cell Migration

Hong-Yo Kang*,† and Chih-Rong Shyr‡,§

Contents

I. In

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uateaner foer, Crtmeorm

troduction

Hormones, Volume 85 # 2011

6729, DOI: 10.1016/B978-0-12-385961-7.00007-X All rig

Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine

r Menopause and Reproductive Research, Chang Gung Memorial Hospital-Kaohsihang Gung University, College of Medicine, Kaohsiung, Taiwannt ofMedical Laboratory Science andBiotechnology,ChinaMedicalUniversity, Taichone Research Center, China Medical University Hospital, Taichung, Taiwan

Else

hts

, Ka

ung

ung

130

II. M

olecular Mechanism of Activin Signaling Regulated Cell

Migration

132

A.

S mads-dependent cell migration 132

B.

S mads-independent cell migration 133

III. T

he Role of Activins in the Regulation of Tumor Cell

Migration and Metastasis

134

A.

P rostate cancer 134

B.

B reast cancer 135

C.

C olon cancer 138

IV. T

he Role of Activins in the Modulation of Immune Cell Migration 139

A.

M ast cells 139

B.

M onocytes 140

C.

D endritic cells 141

V. C

onclusion and Future Prospective 142

Ackn

owledgments 143

Refe

rences 144

Abstract

Activins are the members of transforming growth factor b superfamily and act

as secreted proteins; they were originally identified with a reproductive func-

tion, acting as endocrine-derived regulators of pituitary follicular stimulating

hormone. In recent years, additional functions of activins have been discovered,

including a regulatory role during crucial phases of growth, differentiation, and

development such as wound healing, tissue repair, and regulation of branching

morphogenesis. The functions of activins through activin receptors are pleio-

trophic, while involving in the etiology and pathogenesis of a variety of diseases

and being cell type-specific, they have been identified as important players in

vier Inc.

reserved.

ohsiung,

Medical

, Taiwan

129

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cancer metastasis, immune responses, inflammation, and are most likely

involved in cell migration. In this chapter, we highlight the current knowledge

of activin signaling and discuss the potential physiological and pathological

roles of activins acting on the migration of various cell types. � 2011 Elsevier Inc.

I. Introduction

Multicellular organisms require coordinated migration of cells to fulfilltheir needs in development and homeostasis. For instance, the three primi-tive germ layers are first formed during embryogenesis but later reestablishtheir relative positions during gastrulation; both physiological and patho-logical angiogenesis involve the movement of endothelial cells from pre-existing vessels to new locations to form new vessels. On the other hand,tumor metastases can be interpreted as the result of invasion of the malignantcells to either adjacent or distant healthy tissues by means of lymphatic orhematologic spread, followed by extravasation and establishment of newtumor masses. The aforementioned processes are all intimately linked withcell migration, which is achieved via expressions of morphogens to persuadeand guide movements of specific cell lineages down their genetically deter-mined trajectories. Revealing mechanisms responsible for such preciselyprogrammed migration may shed light on developing novel therapeuticdrug targets for disease control.

In contrast to bacterial chemotaxis, themechanisms for eukaryotic cells tomigrate involve multistage signal transduction, in which external chemotac-tic gradients are converted into intracellular signal gradients. The productionof second messengers then begins and signaling cascades are activated, cul-minating in actin polymerization and the reorganization of cytoskeleton(Ribeiro et al., 2003). It is well established that extrinsic stimuli such asgrowth factors are critical in the process of cell migration, where stringentlocal cues such as target cell type, concentrations of soluble factors, and thesurrounding microenvironment may all contribute (Ribeiro et al., 2003).

Activins are members of the transforming growth factor beta (TGF-b)superfamily along with other multifunctional growth factors, comprising asubfamily of dimeric proteins consisting of two activin b units, which arelinked by a disulfide bridge and contribute to cellular activity regulation(Xia and Schneyer, 2009). Activin A is a dimer of two activin bA subunitsand has been identified to participate in a wide range of actions other thanreproduction (Ball and Risbridger, 2001). Homodimer activin B (bB–bB)and heterodimer activin AB (bA–bB) of the subfamily also bind to theirrespective receptors and commence downstream signaling events(Robertson et al., 1992). Despite the fact that these different forms of activins

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are all bioactive, their nature vary, presenting distinct potencies and cellularfunctions, as demonstrated by in vitro assays with diverse cellular endpoints.The bc and bE subunit genes may encode proteins with antagonistic effects,leading to no downstream signaling whenever a homo- or heterodimercontains these subunits (Muenster et al., 2005), whereas each of the bA andbB subunits is capable of dimerizing with a structurally related but largersubunit to form inhibin A and B (de Kretser et al., 2000). The bindingcapacity of activin type II receptor by activins is lessened by binding ofbetaglycan by inhibins, which negatively regulate activities mediated byactivins (de Kretser et al., 2000).

Activins utilize a type I/type II receptor complex for signal transductionas other members of the TGF-b superfamily. Activin type II receptor(ACVR2 or ActRIIA) is a transmembrane protein harboring serine/threo-nine kinase activity for activin A (Mathews and Vale, 1991, 1993). A type IIreceptor of a different kind (ACVR2B or ActRIIB) has also been found(Harrison et al., 2004). Currently, seven type I receptors, activin receptor-like kinases 1–7 (ALK1–7) have been identified within the TGF-b family(Kang et al., 2009b). Type I receptors are no different from type II receptorsin that they also possess the serine/threonine kinase activity; nevertheless,the uniqueness of type I receptors resides in the possession of theGS domain, which precedes the kinase domain and is close to the intracel-lular juxtamembrane regions. ALK4 is known as the activin type IB recep-tor (ACVR1B or ActRIB), while ALK7 is recognized as the activin typeIC receptor (AVCR1C) (Graham and Peng, 2006). Type I receptorsare recruited to the ligand/ActRII complex as soon as binding of activinsto ActRIIA or ActRIIB occurs; this is followed by phosphorylation of GSdomain by ActRII kinases (Graham and Peng, 2006). Subsequently,activated type I receptors phosphorylate activin/TGF-b-specific Smad,Smad2, and Smad3, which interact with the common mediator Smad4 totranslocate into the nucleus for signal transduction initiation (Tsuchida et al.,2009). Although the DNA-binding property is intrinsic to Smads (Massagueet al., 2005), various DNA-binding cofactors, including CBP/p300, TGIF,c-Ski, and Evi-1 (Kang et al., 2009b), associate with Smads in order to fullyactivate the target genes. There is an array of Smad-interacting transcriptionfactors, ranging from members of the basic helix loop helix (bHLH) family,activator protein-1 (AP-1) family, and homeodomain protein family, toforkhead proteins and nuclear receptors (Derynck and Zhang, 2003). Foraiding target gene regulation, additional transcriptional activators andrepressors are also recruited to the Smad complexes once they are activatedby activins/TGF-b signaling. These characteristics influence the specificpatterns of transcription according to cell types and explain the level ofcomplexity of activins/TGF-b signaling (Derynck and Zhang, 2003;Massague et al., 2005).

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II. Molecular Mechanism of Activin Signaling

Regulated Cell Migration

A. Smads-dependent cell migration

Activin/TGF-b signaling impinges on the organization of cytoskeletal archi-tecture in a rather complex manner. This action primarily involves the actincytoskeleton and secondarily certain systems of intermediate filaments(Zhang et al., 2005). By targeting actin cytoskeleton, it probably aims at aminimum of two interconnected physiological manifestations: First, it facil-itates cell motility, the prerequisites of which are the altered architecturalarrangement and the remodeling of the extracellular matrix to which the celladheres and migrates on. Second, the signaling alters the global architectureof the cell with further impact on its differentiation and proliferation.

For those epithelial cells undergoing epithelial–mesenchymal transition(EMT), the alteration in cellular plasticity is particularly salient, whichinvolves a modification in their differentiation program that increases thetendency for migration; this is essentially associated with the movements oftissues in the embryo and with tumor invasion and metastasis (Ball andRisbridger, 2001; Mercado-Pimentel and Runyan, 2007). During EMT,the intermediate filament system of cytokeratins exchanges to new cytoker-atins and to a vimentin-based skeleton. These changes are functionallyassociated with induced cell mobility toward either a chemotactic gradientof TGF-b ligands or other member of TGF-b members. In the process ofmesenchymal differentiation, Smad and the interacting transcription factorscooperate to provoke the expression of vimentin genes (Wu et al., 2007). Asa result of vimentin synthesis, a new cytoskeleton of intermediate filaments,alpha-smooth muscle actin, and tropomyosins contributes to the assemblyof new actomyosin networks that promote cell motility (Moustakas andHeldin, 2008). In addition, bone morphogenic protein (BMP) receptorcomplex recruitment and BMP-specific Smad signaling are activated andthe new actomyosin networks are established upon myosin X synthesis inthe filopodia of migrating cells (Moustakas and Heldin, 2008).

Smad proteins are considered together with the regulation of actindynamics and the modulation of Rho family GTPases, as exemplified bythe interaction between Smad7 and ALK5 in prostate cancer cells, theformer being inhibitory in nature and functions as a permissive factor forthe activity of Cdc42 to be enhanced after its recruitment by ALK5 as anadaptor (Edlund et al., 2004). The role of Smad3 in EMT has been exten-sively reviewed recently (Xu et al., 2009; Zavadil and Bottinger, 2005).Consistent with these observations, kidney-derived primary tubular epithe-lial cells from Smad3 knockout mice are unable to enter EMT due to afailure of induction of vital regulators of transcription by TGF-b (Zavadil

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et al., 2004). Smad3-dependent EMT is blocked consistently by inhibitorySmad7 overexpression in vitro; this applies to pigment epithelium in theretina (Saika et al., 2004) and the epithelial cells of the mammary gland(Valcourt et al., 2005). EMT may also engage in the model of tumorprogression, with Smad2 and/or Smad3 being implicated as the criticalplayers, since the formation of metastasis and EMT mediating effects areattributed to the cooperation between Smad2 and H-ras (Oft et al., 2002).In addition, overexpression of Smad2 and Smad3 resulted in augmentedEMT in a mammary epithelial model (Valcourt et al., 2005). These studiessupport that receptor-regulated Smads may have a central role in tumorprogression and metastasis-associated EMT of TGF-b-dependent cells.

B. Smads-independent cell migration

Intracellular pathways that are independent of Smad are also regulated, liketheir Smad-dependent counterparts, by the signaling through activin recep-tor complex upon ligand binding (Derynck and Zhang, 2003). p38 mito-gen-activated protein kinase (MAPK), MAPK extracellular signal-regulatedkinases (ERK) 1 and 2, and c-jun N-terminal kinase ( JNK) functionspecifically in their respective cell types and are downstream of activinsignaling (Bao et al., 2005; Giehl et al., 2007); for instance, ERK1/2 activatesthe expression of tyrosine hydroxylase as a result of activin acting synergis-tically with basic fibroblast growth factor. Pituitary transcription factor Pit-1is downregulated by activin via a pathway that is dependent on p38 MAPK,yet the presence of Smad is dispensable (de Guise et al., 2006). In the classicalWnt signaling pathway, the action of the coactivator ActRIB/Smad2 isindependent of Smad4. Tcf4, b-catenin, and the coactivator p300 first cometo close proximity with Smad2 after its activation, and then histone acetyl-transferase activity that resides in p300 allows transcriptional enhancementof b-catenin/Tcf4 via physical interactions (Hirota et al., 2008). On top ofSmads signaling, current evidences incline toward the idea that Rho-GTPases and MAPK are also active in the activin-induced cell migration.The critical steps in activin-induced epithelial cell signal transductionsoccurring independent of Smad4 are ERK 1 and 2 and JNK activation. Inactivin-induced, Smad4-independent cell migration, attention has beengiven to the association of focal complexes with ERK and JNK, whichmay then exert significant effects along with the activated kinases. UnlikeRac and Cdc42, activation of RhoA can be achieved by activins andconsequently leads to JNK and transcription factor c-Jun phosphorylationby MEKK1 (Zhang et al., 2005). p38 activity in keratinocytes from wild-type mice can be triggered by activin via a RhoA-independent pathway;however, such activity cannot be provoked in MEKK1-deficient mice.With their independence from Smad activation in mind, transcription-dependent migration of keratinocytes due to activin stimulation still

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requires both p38 and MEKK1-mediated JNK activity (Zhang et al., 2005).The complexity of activin signaling is further emphasized by the regulationof focal contact turnover by recruited activated ERK and JNK and thereorganization of the dynamic cytoskeleton during activin-induced cellmigration, which simultaneously widens the spectrum of activin-mediated,Smad4-independent cellular events participating in the course of action ofcell migration.

III. The Role of Activins in the Regulation of

Tumor Cell Migration and Metastasis

The establishment of metastatic tumors is like tumorigenesis itselfwhere a multistage process is necessary. The journey taken by the neoplasticcells from the primary tumormass to the distant organs includes intravasationinto, and survival within, the circulation, arrest, and extravasation into thesecondary location; the maintenance of new colonies may be affected by thesuccessful commencing and sustaining of growth and reinitiation of angio-genesis (Chambers et al., 2002). Therefore, only a minute amount of malig-nant cells leaving the primary tumor form metastatic mass eventually(Chambers et al., 2002). Genetic alterations is one of the fundamentalrequirements in the metastatic course, which pave the way for numerouschanged cellular functions shown in both the malignant cells and the hosttissues; these include the regulation of cell–cell adhesion, motility control,and interactions with the extracellular matrix ( Jacks and Weinberg, 2002).It is definitive that such genetic alterations may directly vary the expressionpatterns for certain genes; however, our knowledge to date with regard tothe essence of tumor cell migration based on activin actions is still at itsinfancy.

A. Prostate cancer

While activin A has been previously identified to inhibit the growth ofprostate cancer cells and induces cell-cycle inhibitors such as p27 (Careyet al., 2004), the positive correlation between elevated serum activin A andprostate specific antigen (PSA) levels and increasing Gleason score inpatients with bone-metastasizing prostate cancers is also well-documented(Incorvaia et al., 2007; Leto et al., 2006). The molecular mechanism under-lying these two apparently paradoxical effects of activin A and how activinA influences the progression of prostate cancer with bone metastasis remainsunclear. Based on our recent findings, we found that not only the expressionof activin A is significantly increased in cancer biopsies with a bone meta-static propensity, but also activin A is a key factor promoting cancer cell

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migration to bone matrix produced by osteoblasts. We further discoveredthat activin A activates the androgen receptor (AR) function by modulatingAR mRNA transcription and nuclear translocation through the Smadpathway, and that reduction of AR expression severely impaired the abilityof activin A-treated cancer cells to migrate to bone matrix (Kang et al.,2009a). Hence, a model illustrating the role of activin A in prostate cancermetastasis is hypothesized (Fig. 7.1), in which two phases of downstreamconsequences of activin A’s action on prostate cancer cells may be char-acterized: (1) It increases PSA expression, cell migration, and adherence tobone matrix; these take place within a few hours of low dose activin Asignaling and are counted toward short-term changes. (2) It triggers growtharrest for prostate cancer cells with altered cell morphology, and recruitmentof cancer cells to bone lesions, with formation of osteoblastic matrix. Theseeffects occur under prolonged treatment of activin A and are perceived aslong-term changes. Among the short-term effects, AR activation throughthe Smad-dependent pathway is essential for cell motility enhancement. Atthe molecular level, activated AR has been shown to utilize the Src-FAK-PI3 kinase-Cdc42/Rac1 cascade to mediate its cytoskeletal rearrangingeffects (Castoria et al., 2003). A number of cell surface proteins and proteinsresponsible for cell adhesion and migration, such as ezrin, integrin, andmatrix metalloproteinase-2 (MMP-2), also demand AR for expression(Chuan et al., 2006; Nagakawa et al., 2004). Thus, following our evidencespresented together with reports from others, it is conceivable to believethat prostate cancer cells’ metastatic predilection is linked with intrinsicallydetermined gene expression, which requires strict synchronization ofdifferent signaling complexes and is susceptible to activin-mediated modu-lation through a pathway which may be put forth by the cooperationbetween AR and Smad proteins (Carey et al., 2004; Kang et al., 2001,2002). On the other hand, ALK2-mediated phosphorylation of endoglin,a transmembrane glycoprotein that acts as a TGF-b co-receptor, hasbeen reported to contribute to the regulation of prostate cancer cell migra-tion through Smads-independent pathways (Craft et al., 2007). Thepresent incomplete understanding of activin signaling on cell migrationnecessitates future work to dissect the involvement of activins/AR axisin the Cdc42/Rac1 polarity complex or other cascades related toezrin, integrin, and MMP-2 to influence the motility of malignant prostatecells.

B. Breast cancer

During the first week of lactation, activin A and its binding protein,follistatin, are found in human milk. In both primary and metastatic breastcarcinoma, the gene expression of activin A subunit is more prominent

Neuronendocrinecells

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Metastatic prostatecancer cells

Figure 7.1 Schematic illustration of activin A actions on the progression of prostatecancer. (1) Activin A is highly expressed in prostate cancers with metastatic capacity. (2)Activin A may be secreted as a paracrine factor to adjacent cells and blood vessels. (3)Activin A may induce the arrest of prostate cancer cells. (4) Activin A results in theexpression of AR being upregulated by the cancer cells and promotes cell migrationinto the bone microenvironment. (5) Activin A is capable of mediating the adhesion ofmetastatic cancer cells at or near the cancer–bone interface. (6) Activin A is secreted byosteoblasts; this may result in further activation of metastatic prostate cancer cells. Bluenumbers in the diagram illustrate how activin A works in our model, while graynumbers represent how activin A derived from the cancer and osteoblast cells maydrive a vicious cycle of cancer metastasis.

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compared to that of healthy tissues. When cell homogenates from breastcancer tissue are examined, the concentration of dimeric activin A is doublethe amount of that recorded from surrounding normal tissues. The preciserole of activin A on breast cancer cells is so far waiting to be elucidated, and

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there is little understanding as to how it may entail these phenomena.Activins may suppress the growth of malignant cells, at least in the earlystages of cancer progression. Both Cripto silencing and FLRG silencingtechniques augment activin signaling and retrain the advancement of breastcancer cells (Adkins et al., 2003; Razanajaona et al., 2007). The responsive-ness of breast cancer cells to activin A treatment can be divided into twoclasses depending on their expressions of estrogen receptors (ER). Previousstudies revealed that the growths of cell lines that are deficient of ERexpression were not inhibited by activin A; in fact, activin receptor expres-sion in two of the cell lines proved to be low. In contrast, all of those thatwere positive for ER expression demonstrated activin A-dependent inhibi-tion. (Kalkhoven et al., 1995). A conclusion drawn from the same studyclaims that the accentuated malignancy in ER-negative breast cancer cellsrelative to their ER-positive counterparts may be attributed to the poorresponsiveness of activin signaling in the former (Kalkhoven et al., 1995).Nevertheless, it is too early a stage to assign activin with a cancer repressionidentity, and it is still uncertain in terms of the causative factors in either ERpositive or negative cells of breast cancers taking the discrepancy in activinA actions into account. For breast and prostate cancer patients in later stagesof disease, a remarkable elevation in serum activin concentration wasobserved and similarly, greater activin A level was evident in patients testedpositive for bone metastasis, yet only the correlation between the number ofbone metastases and activin A was considered significant (Leto et al., 2006).Interestingly, a prominent decline in activin A was indisputable during eachof the first two days after tumor resection (Leto et al., 2006). Experimentalinhibition of TGF-b reduced metastasis to various organs, including lung,liver, and bone (Ehata et al., 2007; Ogino et al., 2008; Talmadge, 2008), andthe sensitivity of mammary epithelial cells to TGF-b stimulated migrationwas heightened and elicited by a crucial step which appears to be thephosphorylation of Smad1/5 (Giehl et al., 2007). Smad pathway is not theonly signal transduction pathway identified that participates in the TGF-b-directed cell migration of epithelial breast carcinoma cell lines, JNK, RhoA-GTPase activation, and the ERK 1 and 2 are also implicated (Giehl et al.,2007). Chemical inhibition targeting on ALK4 is a therapeutic strategyshowing promising results, likewise similar effectiveness has also beenobserved in the model of in vivo bone metastasis (Ehata et al., 2007;Halder et al., 2005; Hjelmeland et al., 2004). Together these favor theidea that activins are associated with the pathogenesis of bone metastasis,and provides the rationale for using these cytokines for target therapyaiming primarily at preventing movement of malignant cells, hence combatbone metastasis. Another valuable aspect may be the use of activins as noveldiagnostic markers for metastatic bone diseases.

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C. Colon cancer

Microsatellite instability is one of the core genetic abnormalities found incolon cancers; 58% of 46 such cancer cell lines were identified to possessmutant ACVR2 ( Jung et al., 2007); the speculation of ACVR2 mutationbeing involved in colon carcinogenesis was further stressed by a separatestudy, showing an occurrence of mutations in the same gene, due to aframeshift in exon 10 in all 18 cases, with a proportion as great as 92% out ofa total of 24 colon cancer cell lines and xenografts with high-frequencymicrosatellite instability (Plevova et al., 2004). In specimens for primarycolon cancers, a comparable high rate of mutation was found paralleling theloss of ACVR2 protein expression in the majority of colon cancer cases( Jung et al., 2006). In order to scrutinize the mechanisms and cellular effectsof activin signaling in colon cancer, and to dissect possible new ACVR2signaling transmission routes, ACVR2 function was rescued in two studiesby cellular transfections, where a wild-type ACVR2 was introduced intotumor cell lines with high-frequency microsatellite instabilities that harborthe ACVR2 mutations. Under these circumstances, activin allowedACVR2 protein to form a complex with ACVR1; subsequently, phos-phorylated Smad2 was presented in the nucleus and activin-specific genetranscription was initiated. While reduced growth and S phase along withenhanced cellular migration were observed following activin treatment inthe manipulated cells, small interfering RNA of ACVR2 reversed theseeffects (Deacu et al., 2004; Jung et al., 2007). Certain genes are thought tocontribute to cell growth control and carcinogenesis, including the AP-1complex genes JUND, JUN, and FOSB, and together with the members ofthe small GTPase signal transduction family, RHOB, ARHE, and ARHG-DIA, their expression is amplified due to ACVR2 persuasion, as exhibitedby the microarray-based differential expression analysis. Intriguingly, theoverexpression of these genes coincides with TGF-b receptor 2 (TGFBR2)activation (Deacu et al., 2004). Thus, as one appreciates the analogousfunctional styles between activin and TGF-b signaling systems, it is notsurprising for one to start assessing the possibility that activin signal mayfunction as an alternative route through which the effectors of the TGF-bpathway are activated, with the phosphorylation of Smads being no excep-tion. Regulation of activin-mediated responses may participate in thepathogenesis of high-frequency microsatellite instability colon cancers,considering its growth-restrictive and migration promotion effects akin toTGF-b in the colon cancer scenario. Together, the contribution of theactivin signaling cascade to malignancy requires further evaluation to iden-tify the synergies and differences to other members of the TGF-bsuperfamily.

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IV. The Role of Activins in the Modulation of

Immune Cell Migration

The migration of immune cells is critically important for the protec-tive immune responses to tissues, which mainly involves the positioning ofimmunocompetent cells from bone marrow to the site of inflammation.Inflammation due to tissue damage or infection induces the release ofcytokines and inflammatory chemoattractants from distressed stromal cellsand sentinel cells, such as mast cells and macrophages, which then regulateimmune cell migration by controlling the expressions of adhesion moleculesfor the trafficking of immune cells to the inflamed or damaged tissues(Stupack et al., 2000; Swart et al., 2005). The role of activins in thefunctioning of immune cells remained unclear. Recently, it is beginningto be revealed that activin A could act as a cytokine to regulate immune cellactions, including cell migration.

A. Mast cells

Mast cells (MCs) are derived from multipotent hematopoietic progenitorsin the bone marrow, involved in immediate hypersensitivity and chronicallergic reactions that can contribute to asthma, atopic dermatitis, and otherallergic diseases (Okayama and Kawakami, 2006). MC migration is inducedby various cytokines and chemokines to the sites of inflammation (Stupacket al., 2000), while activin A has autocrine effects on MCs. The induction ofthe activin bA gene in human MCs is stimulated by phorbol 12-myristate13-acetate (PMA) and calcium ionophore (A23187; Cho et al., 2003),whereas the activation of the activin bA gene is achieved by the activationof JNK and p38 kinase through the calmodulin pathway in MCs (Funabaet al., 2003a). The expression of activin A may in turn modulates thefunction of MCs by the upregulating mouse mast cell protease-6(mMCP-6), which is expressed in differentiated MCs (Funaba et al.,2005). With the evidences provided by the modulation of MC responsesin experimental Smad3 depletion (Funaba et al., 2006), it is believed thatSmad3-mediated signaling is essential for maximal cell growth in MCs.Activin A is present in murine bone marrow-derived, cultured mast cellprogenitors (BMCMCs) expressing gene transcripts for molecules involvedin activin signaling, suggesting that BMCMCs could be the target cells ofactivin A. Treatment of activin A inhibited cell growth of BMCMCs in adose-dependent manner and caused morphological differentiation to upre-gulate the mRNA of mouse mast cell protease-1 (mMCP-1), a markerenzyme of mature mucosal MCs. Activin A showed significant activity ininducing the migration of BMCMCs; the optimal concentration for

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maximal migration was 10 pM, which was much lower than the concen-trations required for inhibiting both cell growth and the activation of themMCP-1 gene. Activin A secreted from activated immune cells recruitsMC progenitors to sites of inflammation and that with increasing activin Aconcentrations, the progenitors differentiate into mature MCs. Thus, acti-vin A may positively regulate the functions of MCs as effector cells of theimmune system (Funaba et al., 2003b). The migratory response is probablymediated through its interaction with the TGF-b serine/threonine type Iand II receptors in order to be expressed in the cells. This is supported by theevidence that TGF-b isoforms are highly potent chemotaxins for humanMCs and can play an important role in the recruitment of MCs in inflam-matory reactions (Olsson et al., 2000). Activin A enhances the transcriptioninduced by Microphthalmia-associated transcription factor (MITF)-M andMITF-E, although MITF-mc blocked activin A-induced transcription ofplasminogen activator inhibitor-1 (PAI-1), suggesting that discrete regula-tions of the plasminogen activator system occur in a cell type-specificmanner (Murakami et al., 2006).

B. Monocytes

Monocytes are equipped with phagocytic activity and the ability to differ-entiate into antigen-presenting cells to be involved in both innate andadaptive immune responses. Inflammatory cytokines play a role in regulat-ing where and how do monocytes migrate. The expression of activin A inmonocytes is highly regulated by inflammatory cytokines and glucocorti-coids through a complex network of mechanisms (Abe et al., 2002; Dolteret al., 1998). Activin A expression is also stimulated by bacterial lipopoly-saccharide (LPS) through protein kinase C-dependent transcriptional regu-lation (Eramaa et al., 1992), and the fact that activins increased themigrational activity of monocytes suggested a possible involvement ofactivins in regulating cell-mediated immune function (Petraglia et al.,1991). On the other hand, the proinflammatory cytokines such as IL-1,TGF-b, IFN-g, IL-8, and IL-10 also markedly enhance the expression ofactivin A mRNA in synoviocytes, suggesting their regulatory role in thecontrol of activin A production in bone marrow stroma and monocytes (Yuet al., 1998). In addition, granulocyte–macrophage colony-stimulating fac-tor (GM-CSF), glucocorticoids, or all-trans-retinoic acid were demon-strated to modulate the production of activin A by human monocytes( Jaffe et al., 1995; Yu et al., 1996). Activin A inhibits the production ofinterleukin-1beta (IL-1b), a potent proinflammatory cytokine, andenhances the production of IL-1 receptor antagonist at the posttranscrip-tional level to act as an anti-inflammatory cytokine in inflammatory sites(Ohguchi et al., 1998). Activin A also induces TNF-a from monocytes, butin contrast, activin A has no effect on the production of TNF-b or IFN-g,

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both of which are known to be exclusively generated by T cells, indicatingthat activin A only plays a certain role in the physiological functions ofnormal human monocytes (Yamashita et al., 1993). Specifically, activin Ainduces the differentiation of human monocytes into Langerhans cellsduring inflammatory/autoimmune conditions (Musso et al., 2008).

C. Dendritic cells

Dendritic cells (DCs) are derived from both myeloid and plasmacytoid DC(mDC and pDC) precursors. mDC precursors migrate to inflamed tissues inresponse to inflammatory chemokines and are then remobilized to regionallymph nodes, but pDC precursors transmigrate directly to regional lymphnodes via high endothelial venules (Yoneyama et al., 2005).

DC acts as a source of activin A in vivo, monocyte-derived DC (Mo-DC)releases abundant levels of activin A during the maturation process induced bytoll-like receptor (TLR) agonists, bacteria (Bartonella henselae, Salmonella thy-phimurium), TNF, and CD40L (Scutera et al., 2008). Furthermore, activin A isalso induced in monocyte-derived LC and in blood mDC by LPS and/orCD40L stimulation, but not in blood pDC following stimulation with influ-enza virus. Activin A production by DC is selectively downregulated by anti-inflammatory molecules such as dexamethasone or IL-10. Neutralization ofendogenous activin A using its inhibitor follistatin, or the addition of exoge-nous activin A during LPS treatment does not affect Mo-DC maturationmarker expression, cytokine release, or allostimulatory function. However,Mo-DCmaturedwith LPS in the presence of exogenous activin A displayed ahigher FITC-dextran uptake, similar to that of immature DC (Scutera et al.,2008). Moreover, activin A promoted monocyte differentiation to DC andreversed the inhibitory effects of IL-6 on DC differentiation of monocytes.These findings demonstrate that activinA is released by different subsets ofDC,and it is a cytokine that promotesDCgeneration, affecting the ability ofmatureDC to take up antigens (Ags; Scutera et al., 2008).

Human Mo-DCs, the CD1c(þ) and CD123(þ) peripheral blood DCpopulations express both activin A and the type I and II activin receptors,rapidly secrete high levels of activin A after exposure to bacteria and specificTLR ligands, suggesting that activin A has potent autocrine effects on thecapacity of human DCs to stimulate immune responses (Robson et al., 2008).Blocking autocrine activin A signaling in DCs using its antagonist, follistatin,enhanced DC cytokine (IL-6, IL-10, IL-12p70, and TNF-a) and chemokine(IL-8, IL-10, RANTES, and MCP-1) production during CD40L stimula-tion, but not TLR-4 ligation (Robson et al., 2008). Activin A induces thedirectional migration of immature myeloid dendritic cells (iDCs) through theactivation of ALK4 and ActRIIA receptor chains by the selective and polar-ized release of two chemokines, namely, CXC chemokine ligands 12 and 14through phosphatidylinositol 3-kinase gamma (Salogni et al., 2009).

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V. Conclusion and Future Prospective

Although activins had been known as critical factors to stimulatefollicle-stimulating hormone production from the anterior pituitary sincethe 1980s, an important role in cell migration processes for these proteinshas only emerged more recently. In this chapter, we summarize the currentknowledge of activin signaling and discuss the potential physiological andpathological roles of activins in various cell types during cell migration(Table 7.1). The significant elevation of the level of activin A in theserum of patients correlated with clinically evident prostate cancer meta-stases and PSA levels is particularly noteworthy, placing it as one of theearliest factors in a systemic cascade of cancer progression events. Nonethe-less, its involvement in other tumor diseases like breast and colon cancerspoints to a pivotal function in all metastatic processes and also in associatedpathological migration events. Further delineation of this group of proteincomplex during migration processes will allow their evaluation as potentialdiagnostic measures or therapeutic targets. Preliminary assessment of acti-vins in various cellular migration pathologies may certainly be important,but a more thorough testing of their usefulness in monitoring and treatingmigration-related diseases such as cancer metastasis is warranted. It could besuccinctly summarized as when, where, how, and why in terms of futuredelineation of the basic biology of these proteins in migration processes.The likely cellular timing for activin A-exerted effects is of prime impor-tance particularly during migration processes, as we have argued that thealtered cell behavior is likely to be solely accounted for by the rapid action ofactivin A, yet the more prolonged effects of activin A might also berequired. Which cell types are influenced by these two phases and whethereach phase takes place on distinct populations or has a more global impact isthe subject of ongoing investigation. The mechanism by which activin Alevels are elevated during migration processes is the second question, and itis a fundamental series of cellular and tissue events intimately linked with thetiming of action of activin A. It is also of great interest to systematicallyidentify activins’ target genes during migration processes, such as via micro-array methods. This could help us to better understand the molecular basisof activins’ function and to design new strategies to combine antagonistswith other drugs. For example, various strategies have been designed for theinhibition of activin signaling through receptors and soluble forms of theextracellular domains of activin receptors. Its natural binding protein, fol-listatin, and related ligand binding proteins, chemical kinase inhibitors foractivin receptors, and siRNAs either for ligand or signaling moleculesinterfering with activin signaling, have also been suggested. Once promisingproteins or chemicals targeting activin signaling are discovered, methods of

Table 7.1 Summary of activin A action on various types of cells

Cell types Cell name Activin A actions References

Cancer

cells

Prostate

cancer cells

Reduce cell growth;

promote cell migration

and adherence to bone

matrix; positively

correlate with bone

metastasis

Carey et al. (2004),

Kang et al. (2009a)

Breast cancer

cells

Reduce cell growth;

positively correlate with

bone metastasis

Leto et al. (2006),

Razanajaona et al.

(2007)

Colon cancer

cells

Reduce cell growth and S

phase along with

enhanced cellular

migration

Deacu et al. (2004),

Jung et al. (2007)

Immune

cells

Mast cells Inhibit BMCMCs’

growth; induce

BMCMCs’ migration

Funaba et al. (2003b,

2006), Olsson et al.

(2000)

Monocytes Induce cell migration and

differentiation; enhance

cytokine production

Ohguchi et al. (1998),

Petraglia et al.

(1991)

Dendritic cells Enhance maturation;

promote differentiation;

induce migration

Salogni et al. (2009),

Scutera et al. (2008)

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drug delivery are important issues for effective treatment. The final ques-tion, “why,” has produced some tantalizing hints, particularly from thefindings that the dysregulation of activins may affect functions of gonadsand adipose tissues. What this ultimately means in terms of the roles that theactivins play in migration processes is yet to be deciphered, but the thera-peutic interventions targeted to signaling through activin receptors mayprovide novel strategies for the development of effective treatments againsta variety of diseases.

ACKNOWLEDGMENTS

We apologize to the many researchers whose work could not be cited because of spacelimitations or was only cited indirectly by referring to reviews or more recent papers. Thiswork was supported by grants CMRPD 87041, CMRPG 83021, and CMRPD 83038 fromChang Gung Memorial Hospital and NMRPD 140543 (NSC 94-2312-B-182-054) fromthe National Science Council to Dr. Hong-Yo Kang.

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