Lrukemia and Lymphoma, 2000, Vol. 38(3-4), pp. 265-210 Reprints available directly from the publisher Photocopying permitted by license only

0 2000 OPA (Overseas Publishers Association) N.V. Published by license under

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The CD9 Molecule on Stromal Cells SHIN-ICHI HAYASHIa*, KENSUKE MIYAKEb and PAUL W. KINCADE‘

aDepartment of Immunology, School of Life Science, Faculty of Medicine, Tottori University, Yonago, Tottori, bDepartmeni of Immunology, Saga Medical School, Saga, Japan and ‘Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City, OK, USA

(Received October 17, 1999)

Numerous functions have been attributed to CD9 and other members of the transmembrane 4 (TM4) superfamily. CD9 is thought to be involved in cell proliferation, differentiation, motil- ity and survival. It may also function as part of toxin and virus receptor complexes. Although much remains to be leamed about molecular mechanisms, the molecule associates with sev- eral integrins, small G proteins, MHC class I1 molecules and other TM4 superfamily proteins on a given cell surface membrane. Here, we briefly discuss the CD9 displayed on stromal cells that support hematopoiesis and the potential importance of this molecule to osteoclast differentiation.

Keywords: TM4 superfamily, tetraspanin, osteoclast, hematopoiesis, facilitator

MOLECULAR STRUCTURE OF CD9

CD9 and other transmembrane 4 (TM4) or tetraspanin superfamily proteins are predicted to span cell mem- branes four times with two extracellular loops (TABLE I) (reviewed by Maecker et al.).’ More than 20 TM4 superfamily genes have been identified in

2-4 species as disparate as Schistosoma and mammals. CD9 was first identified on human acute lymphoblas- tic leukemia cells as a 24 kD gly~oprotein.~’~ It has 228 amino acids with one glycosylation site in the first extracellular domain and variable acylation sites (FIGURE l).7-’0 CD9 is known to be expressed in many tissues.” Murine, rat, feline, monkey and bovine homologs have been subsequently cloned.

EXPRESSION AND POTENTIAL FUNCTIONS OF CD9 ON MARROW STROMAL CELLS

The first monoclonal antibody to munne CD9 was established by immunizing with a mesenchymal “stromal cell” clone.12 A large number of cloned stro- rnal cell lines have been derived and many of them were found to express CD9. 12-14 Hematopoiesis is supported within bone marrow by stromal cells, which provide hematopoietic progenitor cells with a scaffold for cell growth and differentiation, by secret- ing a number of hematopoietic growth factors and chemokines as well as by expressing cell adhesion molecule^.^^ Oritani et al. utilized long term bone marrow cultures to implicate stromal cell CD9 in myelopoiesis. l2 While a CD9 specific antibody

* Communicating author: Department of Immunology, School of Life Science, Faculty of Medicine, Tottori University, 86 Nishi-machi, Yonago, Tottori 683-8503, Japan. Phone: +8 1-859-34-8269 FAX: +8 1-859-34-8272 e-mail: shayashi@grape.med.tottori-u.ac.jp

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SHIN-ICHI HAYASHI ef al.

FIGURE 1 A predicted structure of the human CD9 molecule. Cir- cles show each amino acid, and purple (bolded) circles indicate res- idues conserved among TM4 superfamiy A putative glycosylation site is marked as a closed circle (see Color Plate I11 at the back of this issue)

blocked the formation of myeloid cells in “Dexter” type cultures, there was no influence of the same rea- gent on B lineage lymphocyte formation in “Whit- lock-Witte” type cultures. Although myeloid progenitor cells express CD9, the antibody appeared to influence them indirectly via ligation of the CD9 expressed on stromal cells.12 A more recent study exploited the multipotent EML-C 1 hematopoietic cell line and again implicated stromal cell CD9 in hemat- opoiesis.16 Exposure of stromal cells to a CD9 anti- body suppressed EML-C1 cell growth, blocked erythroid differentiation and maintained the EML-C 1 cells in an undifferentiated state. CD9 antibody treat- ment of stromal, but not EML-C1, cells also aug- mented cell adhesion and cobblestone formation, suggesting that it may deliver a pro-adhesive signal. Candidate adhesion molecules associated with CD9 on stromal cells include the pl integrin chain andor an unknown 100 k~ protein.16

TABLE I Members of the TM4 Superfamily

Mammals: CD9 (MRP- 1 ) CD37 (MB-I) CD53 (0x44) CD63 (ME491)

CD82 (KAIl/R2/C33/1A4)

CO-029 (carcinoma antigen) SAS (sarcoma amplified sequence) TALLA-l(T-ALL-associated antigen I) (A15) Tspan-1, -2, -3, -4, -5, -6 NAG-2 (novel antigen-2) L6 (MM3)

late bloomer (Ibl)

genome project

Sm23, Sj23, Sh23 TE736 and Sj2S family

CD8 1 (TAPA- I )

CD15 l(PETA-3EFA- 1)

Drosophila:

C. elegans:

Schistosomas:

PARTICIPATION OF STROMAL CELL CD9 IN OSTEOCL ASTOGENESIS

Osteoclasts are multinuclear, macrophage-like cells that are essential for maintenance of normal bone density and do so by opposing the bone depositing function of osteoblasts. l7-I9 The formation of mature osteoclasts capable of bone resorption is critically dependent on stromal cells. Interestingly, bone form- ing osteoblasts have many features in common with hematopoiesis supporting stromal cells. l8 There has been exciting recent progress in understanding bone physiology and we will speculate that CD9 regulates the function of some key molecular mediators. Osteo- clasts can differentiate from hematopoietic stem cells placed in co-cultures initiated with cloned stromal cells along with la,25-dihydroxyvitamin D3 [ 1 a,25-(OH),D3] .20 Antibodies were effectively uti- lized in that model system to implicate CD9 in osteo- clastogenesis. I4Both osteoclast precursors and the stromal cells that support their maturation express CD9.14321 However, the inhibitory effect of anti-CD9

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CD9 ON STROMAL CELLS 267

antibody was directed to the stromal cells rather than to osteoclast precursors. l4

Osteoclastogenesis is now known to be regulated by a number of molecules and any one of them might be influenced by ligation of stromal cell CD9. Macro- phage colony-stimulating factor (M-CSF) is essential for osteoclast differentiation and M-CSF deficient mice are osteopetrotic.22However, the production of M-CSF transcripts by the ST2 stromal cell line was unaffected by exposing the stromal ceIls to CD9 anti- body. l4

Osteoclast differentiation factor (ODF: also named OPGL, RANKL, TRANCE) represents another important stimulus for osteoclast maturation and another stromal cell It is believed that ODF works in a cooperative fashion with M-CSF. Mice lacking the Odf gene have severe osteopetrosis; lymphopoiesis and lymph node formation are also a f fe~ted .~~The ST2 stromal cell line produces ODF when 1 ( ~ , 2 5 - ( 0 H ) ~ D ~ , parathyroid hormone (PTH), prostaglandins, or interleukin (IL)-1 I are present, thus favoring osteoclast de~e lopmen t .~~ Treatment with 1 a,25-(OH),D3 did not influence CD9 density and ligation of stromal cell CD9 with antibody markedly down-regulated Odf gene expression. l4 This provides one potential explanation of how osteoclast differenti- ation might be modulated.

A major breakthrough in bone studies came with the identification of osteoprotegerin (OPG: also named osteoclastogenesis inhibitory factor).26727 OPG is a soluble decoy receptor for ODE Mice that over-express this molecule have virtually no bone marrow cavities, and Opg deficient mice as well as Odf-overexpressing transgenic mice have osteoclasto- sis, resulting in osteoporosis.28 Although not typical of all stromal cell clones, OPG synthesis by ST2 cells is inhibited under conditions that favor osteoclas- togenesis, i.e. in the presence of 1 u , ~ ~ - ( O H ) ~ D ~ , FTH, prostaglandins, or IL-11 .23327 Addition of CD9 antibody counteracted the tendency of these agents to reduce OPG synthesis (unpublished observation). Continued production of OPG would also create con- ditions unfavorable to osteoclastogenesis and thus increase bone m a ~ s . ~ ~ ” ~ These observations suggest that CD9 may critically regulate bone density in at

least two ways. Substances interacting with stromal cell CD9 within the bone marrow microenvironment would signal reduced production of ODF and contin- ued synthesis of OPG (FIGURE 2). Since ODF pro- duction was not observed in the absence of la,25-(OH),D3, signal(s) delivered via the CD9 mol- ecule could be necessary, but insufficient to induce Odf gene expression. B lineage cells are known to represent another source of ODF, and the augmented 3 lymphopoiesis in IL-7 transgenic mice corresponds to os teopor~s is .~~ This phenomenon might well result from increased osteoclastogenesis driven by the ODF displayed on pre-B cells and thus represents another potential site for CD9 regulation. While CD9 ligation with antibodies does not block the formation of murine pre-B cells in culture, the same treatment is known to deliver a pro-adhesive signal to human pre-B cell^.'^,^^ Therefore, pre-B cells might more readily bind to osteoclast progenitors after CD9 liga- tion, allowing juxtacrine signaling from ODE

ADDITIONAL ROLES FOR CD9

CD9 associates with several integrins and other TM-4 superfamily molecules (TABLE 11). In platelets, CD9 activates phospholipase C indirectly through prior activation of phospholipase A232and associates with a small G protein(s). lo The mernbrane-anchored heparin-binding epidermal growth factor-like growth factor (pro-HB-EGF) is a diphtheria toxin receptor.33 CD9 tightly associates with pro-HB-EGF and acts as a co-factor of pro-HB-EGF in mediating diphtheria toxin sensitivity as well as in juxtacrine growth stimu- latory activity.34 CD9 was previously named motil- ity-related protein (MRP)- 1, because the motility of various tumor cell lines was inhibited by anti-CD9 antibodies.’ In that respect, Cd9 represents a “metas- tasis suppressor gene”. A co-stimulatory signal deliv- ered via CD28 has been well characterized in T cell activation initiated by CD3 ligation. Another co-stim- ulatory pathway that utilizes CD9, rather than CD28 has been reported.35 Recently, Tachibana and Hemler showed that CD9 as well as CD8 1 promote muscle cell fusion and support myotube rnaintenan~e.~~ These

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268 SHIN-ICHI HAYASHI er al.

M-CSF

(Z Maturation C- eoclast

R A M Y L II . .-.... 1-W

FIGURE 2 Potential mechanisms for influence of CD9 molecules on osteoclastogenesis. Osteoclast progenitors differentiate to mature bone resorbing cells under the combined influence of mac- rophage colony-stimulating factor (M-CSF, gene symbol: Csfm) and osteoclast differentiation factor (ODF). This process is opposed by the soluble ODF-decoy receptor, osteoprotegerin (OPG). ST2 stromal cells constitutively produce M-CSF and ODE Addition of Ia,25(OH),D3 to stromal cell cultures induces ODF and sup- presses OPG production, while, M-CSF production is not ~ h a n g e d . * ~ , ~ ~ CD9 on stromal cells physically associates with an unknown molecule(s) (a yellow oval) any member of this complex could interact with counter-receptors on other cells. Experimental ligation of CD9 with an antibody revents Odfgene expression and down-regulation of the Opg (see Color Plate IV at the back of this issue)

observations suggest that CD9 may physically associ- ate with, and modify the functions of, several classes of neighboring plasma membrane molecules. For that rea- son. it has been termed a "molecular facilitator".'

TABLE I1 CD9-associated Molecules

TM4 superfamily: CD63, CD81, CD82, CD151 Integrins: pl (a3pl. a4p1, a6pl) . aIIbp3, aIIb~IIIa , pro-pl Pro-HB-EGF/diphtheria toxin receptor small G protein MHC class I1 CD19 Calnexin Feline immunodeficiency virus Canine distemper virus

Gene targeting thus represents an important tool for evaluating the importance of such molecules and genes for two other members of the TM4 superfamily have already been knocked out. While CD81 is ubiq- uitously expressed in many tissues, null mutant mice only differed from wild-type littermates with respect to humoral immune responses. This is presumably because CD81 physically associates with CD19 on B lymphocytes.37338 Synapse formation is delayed but accomplished by the end of embryogenesis in lbl null mutant Drosophila embryo^.^ Mutants involving such widely expressed molecules often reveal that func- tions of individual TM4 proteins may overlap with other members of the superfamly. There may be com- pensation in many of the tissues where they are nor- mally displayed. Moreover, characteristics of the null mutants are dependent on those of associated mole- cules and it may be that the animals have not been evaluated in the appropriate contexts. In contrast to knockouts, overexpression of a gene can be informa- tive. For example, tumors transfected with Cd9 cDNA lost their highly metastatic proper tie^.^^

CONCLUSION

CD9 and other TM4 superfamily molecules may facilitate the formation of molecular complexes, sta- bilize them and influence their functions. Much of our insight into these processes and mechanisms is lim- ited and based on experiments with antibodies. While antibodies sometimes mimic the inff uence of natural extracellular ligands, treatment with anti-CD9 might block or stimulate normal biological processes for any of several reasons. Ligation of CD9 might deliver a transmembrane signal, disrupt CD9 containing com- plexes, or prevent CD9 from contacting its normal counter-receptor on neighboring cells. We need to be cautious with respect to interpretations, because effects of the antibody can be agonistic andlor antago- nistic. Identification and functional analysis of mole- cules associated with CD9 on stromal cells represents an important goal.

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CD9 ON STROMAL CELLS 269

Acknowledgements

We thank Drs. Takahiro Kunisada, Hidetoshi Yama- zaki, and Toshiyuki Yamane for helpful suggestions.

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