cCAF

9E3/cCAF Manuela Martins-Green* Department of Biology, University of California, Riverside, CA 92521, USA * corresponding author tel: 909-787-2585, fax: 909-787-4286, e-mail: [email protected] DOI: 10.1006/rwcy.2000.10012.

SUMMARY

BACKGROUND

cCAF, a chicken CXC chemokine, is the product of the 9E3 gene and the name reflects its biological properties: chemotaxis and angiogenesis. cCAF is highly homologous to several human CXC chemokines including IL-8 (51%), MGSA (49%), and thromboglobulin (43%). The highest homology resides between the first and fourth cysteines, with the termini of the molecules showing considerable differences. 9E3 gene activation occurs both at the transcriptional and posttranscriptional levels and cCAF is secreted very rapidly as a 9 kDa protein. When cells are cultured on extracellular matrix molecules, cCAF is cleaved in the N-terminus just after secretion to produce a smaller form (7 kDa). Plasmin is an enzyme that cleaves the 9 kDa form of cCAF in the same manner in vitro. cCAF is expressed at low levels by several cell types and can be upregulated by a variety of agents including thrombin, oncogenes, tumor promoters, and wounding. In vivo, cCAF is expressed in endothelial cells of embryonic microvessels; expression disappears upon vessel maturation but can be stimulated by factors known to be angiogenic. cCAF is also highly expressed shortly after wounding and in the course of tissue repair, especially in areas of neovascularization. It is also highly expressed in the stroma of tumors. In the CAM assay, cCAF is chemotactic for monocyte/macrophages and lymphocytes, and is angiogenic, inducing both oriented microvessel growth toward the source of cCAF and sprouting. The angiogenic properties lie in the C-terminus 28 amino acids of the molecule. The biological properties of this chemokine and its patterns of expression in vivo strongly suggest that cCAF plays important roles in wound healing and tumor development.

Discovery The 9E3 gene was one of the first inducible chemokine genes to be discovered. It was identified in 1987 by the Hanafusa group at Rockefeller University (Sugano et al., 1987) when studying v-srcassociated changes in cellular gene expression in order to better understand how this oncogene leads to transformation. Gene expression was analyzed in normal chicken embryo fibroblasts (CEFs) and in Rous sarcoma virus (RSV)-transformed CEFs, using subtraction hybridization techniques to identify genes that were either overexpressed or underexpressed in transformed cells. One particular novel cDNA was expressed at much higher levels in transformed CEFs (Sugano et al., 1987) than in normal CEFs and was given the name of the clone ± 9E3. Independently, Erikson's group at Harvard University isolated the same cDNA and named it CEF4 (Bedard et al., 1987). In the same year, also at Harvard University and using similar technology, Sager's group discovered a gene in tumorigenic Chinese hamster embryo fibroblasts which they termed gro (Anisowicz et al., 1987). Using the cDNA from these tumorigenic hamster fibroblasts as a probe, these investigators also isolated the human GRO gene from a bladder carcinoma cell line (Anisowicz et al., 1987, 1988), which is now known to be identical to the gene for the human melanoma growth stimulating activity (MGSA), a protein discovered in the early 1980s in conjunction with growth stimulation of melanomas (Richmond et al., 1982, 1983). Shortly after the discovery of 9E3 and the GRO/MGSA genes, the cDNA for the closely related neutrophil activating protein (NAP-1)/ IL-8, a protein that is highly expressed in activated

1130 Manuela Martins-Green monocytes (Walz et al., 1987; Yoshimura et al., 1987), was cloned and characterized (Matsushima et al., 1988). Two years later the homolog for GRO in mice was cloned and named KC (Oquendo et al., 1989).

Figure 1 Secondary structure of cCAF predicted by the Garnier method (Garnier, 1978).

Alternative names 9E3 is the gene name given by the Hanafusa group at Rockefeller University (Sugano et al., 1987). CEF4 is the gene name given by the Erikson group at Harvard University (Bedard et al., 1987). cCAF ± the product of the 9E3/CEF4 cDNA ± was given this name by the Martins-Green group at the University of California, Riverside, after the discovery that the protein encoded by this cDNA has chemotactic and angiogenic properties in vivo. cCAF=chicken chemotactic and angiogenic factor (Martins-Green and Feugate, 1998).

Structure The amino acid sequence of cCAF is consistent with some of the classical features of the secondary structure of the chemokine superfamily: an  helix peptide signal sequence, a variable length flexible N-terminus followed by a loop, three -pleated sheets and a Cterminal  helix (Clark-Lewis et al., 1995). This structure folds into a globular protein when the first cysteine forms a disulfide bond with the third cysteine and the second with the fourth. This latter structural feature has been evolutionarily conserved, which strongly suggests importance for at least one major function of chemokines. Figure 1 shows a Garnier plot predicting the secondary structure of cCAF (Garnier, 1978). This secondary structure is a very good predictor of the tertiary structure of this chemokine, as shown below in the discussion of crystal structure.

Main activities and pathophysiological roles When the chorioallantoic membrane (CAM) was used as an in vivo assay, it was found that cCAF is chemotactic for monocyte/macrophages and lymphocytes (Martins-Green and Feugate, 1998). Two days after application of methylcellulose pellets containing 100±300 ng/pellet cCAF to the CAM, both monocyte/ macrophages and lymphocytes were attracted. However, cCAF was not chemotactic for heterophils as might have been expected of a CXC chemokine. Hyperproliferation of the CAM ectodermal cells was also observed and a large number of the fibroblasts became elongated and aligned parallel to each other

beneath the pellets (Martins-Green and Feugate, 1998). Four days after treatment with the protein at these concentrations, the region under the pellet was occupied by large numbers of fibroblasts with abundant deposition of collagen, very similar to the granulation tissue of wounds. In addition, numerous new blood vessels were present in the pellet and in its vicinity. However, at higher concentrations of the protein (500±1000 ng/pellet), no chemotaxis for leukocytes nor any of the other wound healing effects outlined above (hyperproliferation of ectodermal cells, fibroblast alignment, collagen deposition, blood vessel growth towards the pellet) were observed. Rather, abundant sprouting of existing blood vessels was observed at 4 days after application of the pellets containing cCAF. The sum of these observations suggests that at lower doses cCAF has a direct chemotactic effect for monocyte/macrophages and at higher doses it causes sprouting of new blood vessels. Whether the remainder of the wound healing-like effects are entirely a result of induction of secretory products from macrophages or partially due to direct cCAF effects remains to be determined. Further studies have investigated the angiogenic activities of cCAF by determining what part of the molecule is responsible for this function (MartinsGreen and Kelly, 1998). Again the CAM assay was used and showed that low doses of the C-terminal 28 amino acid peptide stimulated oriented blood vessel growth without attracting leukocytes (Martins-Green and Kelly, 1998). At higher doses the peptide stimulated extensive tortuosity and sprouting of the tertiary

9E3/cCAF 1131 Figure 2 Schematic representation of the biological activities of cCAF. cCAF is stimulated, synthesized, and secreted as a 9 kDa molecule. After secretion it can be processed at the N-terminus to a smaller form of about 7 kDa which binds to extracellular matrix (ECM) molecules. Whereas the larger form is chemotactic for leukocytes, the smaller form contains the C-terminus that has the angiogenic properties. Question marks are placed on those functions under current study.

blood vessels. When the peptide was deposited under the skin of the wings of young chicks, there was an increase in the number of microvessels in the muscle tissue at the site of application, without the presence of leukocytes (Martins-Green and Kelly, 1998). The C-terminal peptide recapitulates all of the angiogenic properties of the full cCAF molecule, but does not have chemotactic properties for leukocytes, and it does not cause development of granulation-like tissue. These biological activities are summarized in Figure 2.

GENE AND GENE REGULATION

Accession numbers The gene has not yet been isolated. cDNA: M16199

Regulatory sites and corresponding transcription factors Analysis of the upstream regulatory region of the 9E3 gene using the Transcription Element Search Software

(TESS) assay (Heinemeyer et al., 1998), which recognizes consensus transcription factor-binding elements, identified a variety of elements in the promoter sequence of this gene (Figure 3). These DNA elements are probably responsible for the binding of transcription factors activated by the vast array of stimuli that turn on this gene. Previous studies have been directed toward identification of the DNA elements involved in activation of 9E3 by v-src. The tyrosine kinase activity of this oncogene causes an increase in the levels of the CCAATT-binding factor and this element is responsible for the basal level of transcription (Dutta et al., 1990; Faber and Sealy, 1990). Activation of the 9E3 promoter in tCEFs also requires PDRII/B and AP-1, two other elements close to the TATA box (Dehbi et al., 1992). Mutations in any of these sequences dramatically reduced activation and a single copy of any of these sites in the promoter also had no stimulatory capabilities. However, multiple copies of AP-1 or of PDRII/B (the avian equivalent of the NFB family) (Leonardo et al., 1989) sequences conferred activation of the promoter by v-src to levels above that conferred by the normal sequence (Dehbi et al., 1992; Bojovic et al., 1996). In addition to v-src, the 9E3 gene is stimulated by a variety of agents (see Eliciting and inhibitory stimuli), with thrombin being the strongest natural activator of this gene so far identified. Nothing is known about the transcription factors involved in the stimulation of this gene by most of these agents. Although activation of transcription of this gene by thrombin has only been partially worked out, it involves elements in the DNA other than those involved in v-src activation (Li and Martins-Green, 1998; Li et al., 1998, 1999). This is not surprising since transcription activation by v-src takes hours (Gonneville et al., 1991) rather than minutes as is the case with thrombin (Vaingankar and Martins-Green, 1998) and phorbol esters (Li et al., 1999). Moreover, v-src activation involves PKC, a Ser/Thr kinase (Spangler et al., 1989; Qureshi et al., 1991), whereas activation by thrombin does not involve PKC but instead requires tyrosine kinase activity (Vaingankar and Martins-Green, 1998). Thrombin stimulation of 9E3/cCAF is very rapid and occurs via its proteolytically activated receptor with subsequent transactivation of the EGF receptor tyrosine kinase and of the nonreceptor tyrosine kinase pp60src. Downstream from the EGF receptor, Ras and Raf are involved with subsequent activation of MEK1 and ERK2. Two Elk1-binding elements located between ÿ534 and ÿ483 bp of the promoter are the major thrombin response elements and activation of transcription of 9E3/cCAF occurs via direct activation of the Elk1 transcription factor by

1132 Manuela Martins-Green Figure 3 Schematic representation of the promoter region of the 9E3 gene with consensus-binding elements for known transcription factors. The elements were identified by TESS (Transcription Element Search Software, from Transfac Database http://transfac.gbf.de/). Only the most common consensus-binding elements for stress responses are shown. The multiple stress-response elements indicate tissue- and stimulispecific regulation of this gene. GR, glucocorticoid receptor; AP-1, activating protein 1; Oct-1, conserved octomer-binding protein-1; Ets, E26 specific sequence encoding protooncoprotein; Elk1, Ets-like factor-1; C/EBP, CAAT element-binding protein.

(a)

9E3/cCAF 1133 ERK2. Furthermore, transcription activation of this chemokine gene by Elk1 does not require cooperation with AP-1 (Li et al., 1998; Martins-Green and Li, 1999). The common occurrence of Elk1-binding domains in the promoters of immediate early response genes (such as chemokines) that are activated in response to stress signals, suggests that this transcription factor may be characteristically involved in activation of these genes by stress-inducing agents. Activation of transcription of 9E3/cCAF by phorbol esters (Li et al., 1999) is similar to that of thrombin downstream from MEK1/ERK2, but the signaling pathways that lead to activation of these kinases are different from those stimulated by thrombin upon binding to its receptor. Phorbol esters stimulate this gene via three signaling pathways simultaneously: (i) a small contribution through protein kinase C (the commonly recognized pathway for these tumor promoters); (ii) a contribution involving tyrosine kinases; (iii) a larger contribution via pathways that can be interrupted by dexamethasone. In addition, activation of Elk1 by these signaling pathways is independent of AP-1 and PDRIIB (an NFB-like factor in chickens). The very rapid activation of 9E3 by thrombin is reminiscent of the equally rapid activation of heat shock genes, suggesting that the stress of wounding could be analogous to the stress of heat. When cells are suddenly heated, they activate heat shock transcription factors which bind the heat shock elements in the DNA, causing gene transcription (Morimoto, 1993). The 9E3 gene does not appear to be activated by heat shock, but there may be a similarity in transcription activation mechanisms because activation of the 9E3 gene by thrombin is also detectable within minutes (Vaingankar and Martins-Green, 1998). Therefore, it is possible that chemokines constitute a new class of stress response proteins whose function is to defend an organism against an external threat requiring the coordinated response of many factors and cells. Regulation of 9E3 gene expression also occurs at the level of mRNA stability (Stoeckle and Hanafusa, 1989). In addition to the full-length mRNA, two smaller forms of RNA were found by probing northern blots with cDNA probes. One form hybridized with a 50 cDNA probe and contained the coding region, and the other hybridized with a 30 cDNA probe and did not contain any of the coding region but had the polyadenylation site. These smaller forms were not the result of expression of different genes nor were they the result of differential splicing of the same hnRNA; they were the result of processing of one transcript. The 30 terminus of the 50 RNA

mapped to the same position as the 50 end of the 30 RNA, suggesting that the large transcripts undergo endonucleolytic cleavage at a specific site in the 30 noncoding region. In addition, the smaller forms were always found to be more abundant under conditions that render the full length transcript unstable, suggesting that this endonucleolytic cleavage regulates mRNA stability and therefore is important in posttranscriptional regulation of gene expression (Stoeckle and Hanafusa, 1989).

Cells and tissues that express the gene See Table 1. In normal quiescent chicken embryo fibroblasts (CEFs) or primary avian tendon (PAT) cells, the 9E3 gene is expressed at barely detectable levels, but it is more highly expressed in growing cultures and can be stimulated to still higher levels of expression by the addition of medium containing 5±10% serum (Sugano et al., 1987; Stoeckle and Hanafusa, 1989; MartinsGreen et al., 1991, 1996). Expression under conditions that stimulate growth begins during the G0 !G1 transition of the cell cycle and declines during S-phase (Martins-Green et al., 1991) (Figure 3). RSV (Rous sarcoma virus)-transformed CEFs express the gene constitutively and at high levels. This expression correlates well with the transforming capabilities of the virus; the 9E3 mRNA levels are high in fully transforming mutants of RSV, and low in transforming negative mutants or with virus expressing c-src in place of v-src (Sugano et al., 1987). Activation of the gene requires an active kinase as well as the proper association of the kinase with the cell membrane, because mutants that lack N-terminal myristilation are incapable of stimulating the gene (Sugano et al., 1987). In CEFs transformed with a ts mutant of RSV, upon activation of the pp60v-src kinase, the gene is expressed within 15 minutes (Gonneville et al., 1991) in a biphasic manner, with an early transient phase peaking at 2 hours and a late constitutive period that reaches its maximum at 24 hours (Gonneville et al., 1991). The early expression is the result of mRNA stabilization, whereas the later elevation of expression is the result of transcriptional activation (Gonneville et al., 1991). In normal tissues of newly hatched chicks, 9E3 is expressed and cCAF is produced in connective tissue fibroblasts, tendon cells, and osteoblasts, but not in muscle, bone marrow, or endothelial cells of fully developed blood vessels (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). However, mature

1134 Manuela Martins-Green endothelial cells can be stimulated to produce cCAF by agents that cause inflammation (Martins-Green, 1993). In contrast, after wounding cCAF is more abundant in areas where microvessels are present. It is produced in the endothelial cells of these microvessels, and it is found in extracellular matrix, especially where collagen is abundant (MartinsGreen and Bissell, 1990; Martins-Green et al., 1992). The gene is also upregulated in the cells of the healing epithelium and in the connective tissue underlying this epithelium where interstitial collagen, laminin (Martins-Green and Bissell, 1990; Martins-Green et al., 1992) and tenascin (Mackie et al., 1988) are abundant. cCAF is the first of the chemokines to be found to associate with ECM molecules other than glycosaminoglycans. Because this chemokine is expressed in tissues rich in interstitial collagen, is overexpressed during wound healing where ECM is abundant (Martins-Green and Bissell, 1990; MartinsGreen et al., 1992), and is stimulated in normal cells when they are cultured on interstitial collagen or basement membrane components (Martins-Green et al., 1993), it is likely that interactions with the matrix are of fundamental importance for the function of the protein. Different modes of availability of the two isoforms (see Description of protein) could lead to different functions in vivo (Martins-Green et al., 1996). In areas where tumors are developing, both the mRNA and protein are elevated in the tumor stroma, but not in the tumor cells themselves (Martins-Green et al., 1992). The very high expression of 9E3 in transformed CEFs and the lack of expression in the cells of RSV tumors is difficult to understand. However, these tumors are rhabdomyosarcomas, meaning that they are composed primarily of transformed muscle cells. One possible explanation is that normal muscle cells do not express the 9E3 gene, hence their transformed equivalents may not either. Very little is known about the expression of this gene during embryogenesis. Expression primarily occurs in epidermal cells and in the endothelial cells of young blood vessels, but as development proceeds and the blood vessels mature, cCAF is no longer found in endothelial cells (Martins-Green and Feugate,

1998). Studies are in progress to examine in detail the expression of this chemokine in organ tissues during the various stages of embryonic development.

PROTEIN

Accession numbers Protein sequence: gi211735 PDI: g211736

Sequence See Figure 4.

Description of protein The cCAF chemokine is secreted as a 9 kDa form in both normal and transformed cells. However, when cells are cultured on extracellular matrix molecules, a smaller form of the protein (7 kDa) can be produced by postsecretory cleavage at the N-terminus (MartinsGreen et al., 1996). This smaller form binds to interstitial collagen and basement membrane components. Study of binding to basement membrane components revealed that cCAF binds abundantly to laminin, to a lesser extent to complex proteoglycans, and does not bind to collagen IV (Martins-Green et al., 1996). In addition, this chemokine also binds to tenascin, an ECM molecule abundantly expressed in wounds and tumor stroma, but does not bind to fibronectin and hyaluronic acid. Plasmin is the only enzyme known to cleave cCAF to yield a C-terminus-bearing smaller form of the same size as found when cells are cultured on ECM (Martins-Green et al., 1996). This enzyme is released at sites of wounds and produced in association with tumors, therefore processing by plasmin could be relevant during healing and tumorigenesis. The 9 kDa form exists primarily as monomers in the supernates of cultured CEFs and is stable for a period of 24 hours in normal cultures, but has a

Figure 4 Amino acid sequence. The signal peptide is underlined. The cysteines that contribute to the formation of the two disulfide bonds that are a fundamental characteristic of the chemokine superfamily are in bold and the ELR motif is highlighted. MNGKLGAVLA LLLVSAALSQ GRTLVKMGNE LRCQCISTHS KFJHPKSIQD VKLTPSGPHC KNVEIIATLK DGREVCLDPT APWVQLIVDA LMADAQLNSD APL

9E3/cCAF 1135 half-life of 3 hours in cultures of transformed CEFs. Neither form of the molecule is glycosylated or phosphorylated nor does either bind to heparin with high affinity (Martins-Green et al., 1996). Pulse-chase experiments showed that cCAF is synthesized and secreted very rapidly, in less than 10 minutes (Martins-Green et al., 1996). Therefore, given an appropriate stimulus, the level of cCAF could be very rapidly elevated in vivo, resulting in a similarly rapid biological response. Presumably at least one function of cCAF must require its extremely rapid availability.

Discussion of crystal structure The crystal structure of cCAF is in the process of being determined. However, because cCAF is a CXC chemokine and is highly homologous to human IL-8 (51% of the entire molecule at the amino acid level; 62% from the ELR motif through the fourth cysteine) the crystallographic structure of IL-8 has been used to model the structure of cCAF. Studies using NMR spectroscopy (Clore et al., 1989, 1990) and X-ray crystallography (Baldwin et al., 1991) show that the structure of IL-8 consists of a short, flexible, Nterminus followed by a loop, three -pleated sheets, and a C-terminal  helix. The cCAF structure consists of the same domains, but the N-terminus of the molecule is 10 amino acids longer and the C-terminus is five amino acids longer than the termini of IL-8 (see Important homologies). To model the cCAF structure, the three-dimensional structure of IL-8 was obtained from the PDB database and modified by replacing the amino acids of IL-8 with those of cCAF using the Builder module of the Insight software (BIOSYM, San Diego, CA, USA). The method of Garnier was then used to predict the secondary structures of the N- and Ctermini of cCAF, the regions of the molecule that differ most from the other CXC chemokines (Garnier, 1978) (see Structure). It was found that the first five amino acids of the N-terminus assume an  helix configuration not present in the structure of IL-8. The C-terminus displays an  helix very similar to that of IL-8 and MGSA, with the last five amino acids (not present in the two human chemokines) forming a flexible extension of the helix. This structure was then optimized through energy minimization and molecular dynamics calculations using the Discover module (BIOSYM). After tens of thousands of iterations, the structure shown in Figure 5 was obtained.

Figure 5 Three-dimensional representation of the cCAF molecule. Structure was generated starting from the structure of IL-8 by replacement with the amino acids of cCAF followed by 180,000 iterations to minimize the free energy of the tertiary structure. Note that the long N-terminus (blue) assumes a small  helix and folds back over the body of the molecule. The C-terminus  helix (lavender) similarly folds over the body of the molecule (yellow) such that the last few amino acids align with the sheets (white). (Full colourfigurecanbeviewedonline.)

Important homologies Despite the fact that cCAF is an avian chemokine, the sequence of this protein is more homologous to human IL-8 (51%) than any other chemokine, human or otherwise, with the homology largely confined to the region between the cysteines (Figure 6). It is similarly highly homologous to MGSA (45%), and to -thromboglobulin (43%), two other human chemokines (Stoeckle and Barker, 1990). Functionally, cCAF also resembles many of the CXC chemokines because of its angiogenic properties. However, its chemotactic properties for monocyte/macrophages more closely resemble MCP-1, a CC chemokine that is also chemotactic for monocytes. MCP-1's chemotactic function localizes to the N-terminus (Grewal et al., 1997) where the molecule assumes an  helix structure (Lubkowski et al., 1997). As described above, the molecular modeling studies show that the N-terminus of cCAF assumes an  helix structure much like MCP-1 (Martins-Green and Hanafusa, 1997). IL-8 and MGSA, however, lack this

1136 Manuela Martins-Green structure in their N-terminus and also lack monocyte chemotaxis. In addition to the homologies described above, cCAF shows 67% homology to a newly discovered chicken CXC chemokine (Sick and Staeheli, 1997). Other recently discovered avian chemokines are MIP1 (Petrenko et al., 1995), which shows 57% homology to its human counterpart, and lymphotactin, a

Figure 6 Sequence homology between cCAF, IL-8, and MGSA. Most of the homologies between cCAF and the two human chemokines reside in the core portion of the molecules with the termini showing the least homology. cCAF is 51% homologous to IL-8 and 49% homologous to MGSA.

C chemokine that also displays high homology to its mammalian counterpart.

Posttranslational modifications Only two disulfide bonds.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce RSV-transformed chicken embryo fibroblasts (CEFs) produce high levels of cCAF. Using these cells as a source for this chemokine and employing classical chromatography, the chemokine has been purified and now is available for in vivo assays (Martins-Green and Bissell, 1990; Martins-Green and Feugate, 1998). See also Table 1.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Studies designed to correlate 9E3 expression with mitogenesis and with cell shape changes that occur during transformation led to the identification of a number of transforming viruses and pharmacological agents that stimulate expression of this gene (Barker and Hanafusa, 1990). Transformation with oncogenic viruses causes an upregulation of the 9E3 gene. This Table 1 Summary of 9E3/cCAF expression in vivo Expressed

Not expressed

Overexpressed

Connective tissue fibroblasts

Muscle cells

Wounded tissues, especially in areas of neovascularization and where interstitial collagen is abundant

Osteoblasts

Mature endothelial cells

Tendon fibroblasts

Bone marrow cells

Normal tissues

Young endothelial cells Pathological tissues Tumor cells

In tissues around tumors

9E3/cCAF 1137 is true both for viruses with and without tyrosine kinase activity, suggesting that tyrosine kinases are not necessarily involved in activation of 9E3 by oncogenes (Barker and Hanafusa, 1990). Indeed, it has been shown that v-src and v-fps use a mitogenic PKC-mediated pathway for activation of the 9E3 gene (Spangler et al., 1989; Qureshi et al., 1991). Activation via Ser/Thr kinases is also consistent with stimulation of this gene by agents such as okadaic acid, a nonphorbol tumor promoter that inhibits ser/ thr phosphatases 1 and 2A, and LPS, which causes inflammation and modulates the phosphorylation of one of the substrates of PKC in macrophages (Barker and Hanafusa, 1990). However, vanadate, which is a specific inhibitor for tyrosine phosphatases, also activates 9E3 (Barker and Hanafusa, 1990), indicating that tyrosine kinases can be important in the activation of this gene (Vaingankar and Martins-Green, 1998) (Figure 7). In addition to stimulation by these pathological and pharmacological agents, considerable 9E3 expression is also rapidly induced by wounding (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). Factors released upon wounding which could cause the very rapid stimulation of the 9E3 gene include components of the platelet  granules (such as PDGF, TGF , TGF), other growth factors that are produced locally after wounding (such as EGF, FGF, and bFGF), and the enzyme thrombin. When these factors were tested for stimulation of 9E3 expression, all stimulated expression of the gene to some degree. However, thrombin was the most potent stimulator, causing levels of expression 15 times higher than those in quiescent, confluent CEFs, but comparable to those found in

transformed CEFs (Vaingankar and Martins-Green, 1998), which express the gene constitutively (Sugano et al., 1987; Stoeckle and Hanafusa, 1989; Barker and Hanafusa, 1990). Studies of activation of the 9E3 gene by thrombin have shown that activation by this enzyme occurs very rapidly ± 1 minute exposure of cells to thrombin is sufficient to stimulate this gene. This stimulation is receptor-mediated via the proteolytically activated receptor for thrombin (Vaingankar and Martins-Green, 1998). In contrast to the activation by v-src, which acts via a mitogenic pathway involving the Ser/Thr kinase PKC, activation by thrombin through the proteolytically activated receptor for thrombin is independent of mitogenesis and involves transactivation of the EGF receptortyrosine kinase (Vaingankar and Martins-Green, 1998). The c-src tyrosine kinase is also activated upon thrombin stimulation of CEFs and may be involved in the transactivation event (Vaingankar and Martins-Green, 1998). Because of its stimulation through these independent pathways, inhibition of 9E3/cCAF expression varies depending on the stimulant. The pathways involving tyrosine kinases are blocked by inhibitors of these kinases and of the stress-induced kinase cascade (Vaingankar and Martins-Green, 1998). On the other hand, stimulation via growth-related pathways is blocked by inhibitors of Ser/Thr kinases (Barker and Hanafusa, 1990). In addition, this chemokine can be inhibited by antiinflammatory agents such as dexamethasone (Barker and Hanafusa, 1990; Li et al., 1999). The very fact that the expression of this chemokine can be suppressed by inhibitors of various signal transduction pathways supports our findings (see Regulatory sites and corresponding transcription factors) that the promoter region of the 9E3 gene is rich in a variety of consensus-binding elements for diverse transcription factors.

Figure 7 Schematic representation of the activators and inhibitors of 9E3/cCAF expression.

RECEPTOR UTILIZATION Receptors are not yet known.

IN VITRO ACTIVITIES

In vitro findings Studies with the Boyden-Chamber assay (Barker et al., 1993) demonstrated that cCAF is chemotactic for peripheral mononuclear leukocytes.

1138 Manuela Martins-Green

Regulatory molecules: Inhibitors and enhancers See Eliciting and inhibitory stimuli.

Bioassays used For chemotaxis in vitro, the Boyden-Chamber assay. For chemotaxis and angiogenesis assays in vivo, the chorioallantoic membrane (CAM).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles cCAF may play normal physiological roles in wound healing (see Main activities and physiological roles). The 9E3 gene is expressed at high levels shortly after wounding, expression remains very high during the early inflammatory phase of healing, but after 36±48 hours decreases to a plateau of elevated expression which remains constant throughout granulation tissue formation (Figure 8a) (Martins-Green and Hanafusa, 1997). The very high levels of gene expression found shortly after wounding potentially can be explained by the fact that in culture 9E3 gene expression rises to high levels within minutes after thrombin stimulation of fibroblasts (the cells that in the wound most highly express 9E3/cCAF) (Vaingankar and Martins-Green, 1998). The results on the CAM (see also MartinsGreen and Feugate, 1998; Martins-Green and Kelly, 1998) further suggest that this very high level of cCAF production after wounding is responsible, at least in part, for the chemotaxis of monocytes and lymphocytes that are components of the inflammatory response, and for fibroblasts that are involved in the formation of the granulation (repair) tissue. In vivo, this repair tissue expresses high levels of 9E3, but expression falls off progressively with distance from the wound (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). At the same time, cCAF is found in a similar pattern in association with extracellular matrix, especially with interstitial collagen (Martins-Green and Bissell, 1990; MartinsGreen et al., 1992). The change of level of 9E3 expression (Figure 8a) and initiation of the binding of cCAF to ECM coincides in time with the generation of plasmin at the wound site. Because plasmin processes cCAF at the N-terminus resulting in the smaller

form which binds to collagen, laminin, and tenascin (Martins-Green et al., 1996), it is possible that the coincidence in time of plasmin appearance and cCAF build-up in the matrix represents cause and effect. Furthermore, it is possible that the binding of cCAF to the ECM and production of a persistent gradient of this chemokine might form the basis for the chemotaxis of endothelial cells, because this property of cCAF lies in its C-terminus (Martins-Green and Kelly, 1998). At the same time, in the area close to the wound, the locally elevated level of cCAF could induce sprouting of blood vessels. Based on these findings, we hypothesize that cCAF participates in wound healing in the following ways (Martins-Green and Hanafusa, 1997) (Figure 8b). Wound repair is initiated by the thrombin-stimulated clotting cascade. At the same time, thrombin stimulation of local fibroblasts to express 9E3 leads to secretion of the full-length form of cCAF which contributes to the inflammatory phase of wound healing via monocyte chemotaxis. In the later stages of inflammation, endothelial cells produce plasminogen activator which processes plasminogen into plasmin that in turn cleaves cCAF to the smaller form, rendering it capable of binding to the interstitial collagen being deposited at the site of the wound. As healing progresses and collagen deposition continues within the granulation tissue, its presence not only provides more matrix for cCAF binding but also further stimulates the production of this chemokine. Binding of the smaller form to the matrix provides a persistent gradient for chemoattraction of endothelial cells to the granulation tissue and the elevated level of cCAF in this repair tissue induces sprouting of new vessels that form the microvasculature of the wound bed. The pathophysiological roles of cCAF relate to tumor development. The cells of RSV-induced tumors do not express the 9E3 gene or produce cCAF but the tumor stroma does (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). The time dependence of the expression of the gene after stimulation of tumors by viral injection showed that there is a small elevation of 9E3/cCAF during the early stages of tumor development, but as tumors grow expression increases, reaching high levels during late stages of tumor growth (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). In cases where the immune system overcomes RSV infection, tumors regress, accompanied by a large influx of lymphocytes (Gelman and Hanafusa, 1993). Therefore, it is possible that during the early stages of tumor development the low levels of cCAF attract monocyte/macrophages and lymphocytes, much as was observed in the CAMs treated with low concentrations of cCAF (Martins-Green and Feugate, 1998). If

9E3/cCAF 1139 Figure 8 Model for the mechanisms of action of cCAF in wound healing. (a) Levels of cCAF mRNA after wounding and during healing. Quantification of RNA was performed by microdensitometry. Shortly after wounding the gene is very highly expressed, remains high for 36±48 hours, and then declines to a plateau of lower but persistent expression during granulation tissue formation. The high stimulation correlates with the inflammatory phase of the wound and therefore with the time in which chemotaxis for leukocytes occurs. The lower plateau correlates with granulation tissue formation and therefore with angiogenesis and mitogenesis. (b) A model of activation of the 9E3 gene by thrombin and other wound factors, and how cCAF potentially contributes to wound repair.

1140 Manuela Martins-Green the tumor is aggressive it continues to grow and causes more and more of the surrounding tissue to express the 9E3 gene (Martins-Green and Bissell, 1990; Martins-Green et al., 1992). Developing tumors produce a variety of enzymes, one of which is plasminogen activator (Bell et al., 1990; Schmitt et al., 1995; Torre and Fulco, 1996); in particular it has been shown that this factor is induced by v-src (Bell et al., 1990). As mentioned above, plasminogen activator converts plasminogen (present in the tissues) into plasmin, which could process cCAF to its smaller, matrixbinding form (see Description of proteins). Build-up of a gradient of this chemokine around the tumor could induce angiogenesis. Therefore, at early stages of tumor development, cCAF may be involved in interfering with tumor development, whereas, if the tumor has already gained a foothold over the immune system, cCAF may be playing a permissive role that favors tumor growth by stimulating angiogenesis.

Species differences 9E3/cCAF has not yet been isolated in other species.

References Anisowicz, A., Bardwell, L., and Sager, R. (1987). Constitutive over-expression of a growth-regulated gene in transformed Chinese hamster and human cells. Proc. Natl Acad. Sci. USA 84, 7188±7192. Anisowicz, A., Zajchowski, D., Stenman, G., and Sager, R. (1988). Functional diversity of gro gene expression in human fibroblasts and mammary epithelial cells. Proc. Natl Acad. Sci. USA 85, 9645±9649. Baldwin, E. T., Weber, I. T., St Charles, R., Xuan, J-C., Appella, E., Yamada, M., Matsushima, K., Edwards, B. F. P., Clore, G. M., Gronenborn, A. M., and Wlodawer, A. (1991). Crystal structure of interleukin 8: symbiosis of NMR and crystallography. Proc. Natl Acad. Sci. USA 88, 502. Barker, K., and Hanafusa, H. (1990). Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts. Mol. Cell. Biol. 10, 3813±3817. Barker, K. A., Hampe, A., Stoeckle, M. Y., and Hanafusa, H. (1993). Transformation-associated cytokine 9E3/CEF4 is chemotactic for chicken peripheral blood mononuclear cells. J. Virol. 67, 3528±3533. Bedard, P.-A., Alcorta, D., Simmons, D. L., Luk, K.-C., and Erikson, R. L. (1987). Constitutive expression of a gene encoding a polypeptide homologous to biologically active human platelet protein in Rous sarcoma virus-transformed fibroblasts. Proc. Natl Acad. Sci. USA 84, 6715±6719. Bell, M. B., Brackenbury, R. W., Leslie, N. D., and Dengen, J. L. (1990). Plasminogen activator gene expression is induced by the src oncogene product and tumor promoters. J. Biol. Chem. 265, 1333±1338. Bojovic, B., Rodrigues, N., Dehbi, M., and Bedard, P. A. (1996). Multiple signaling pathways control the activation of the CEF-

4/9E3 cytokine gene by pp60v-src. J. Biol. Chem. 271, 22528± 22537. Clark-Lewis, I., Kim, K.-S., Rajarathnam, K., Gong, J.-H., Dewald, B., and Moser, B. (1995). Structure-activity of chemokines. J. Leukoc. Biol. 57, 703±711. Clore, G. M., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1989). Determination of the secondary structure of interleukin-8 by nuclear magnetic resonance spectroscopy. J. Biol Chem. 264, 18907±189211. Clore, G. M., Appella, E., Yamada, M., Matsushima, K., and Gronenborn, A. M. (1990). Three-dimensional structure of interleukin 8 in solution. Biochemistry 29, 1689±1693. Dehbi, M., Mbiguino, A., Beauchemin, M., Chatelain, G., and BeÂdard, P. A. (1992). Transcriptional activation of the CET-4/ 9E3 cytokine gene by pp60v-srv. Mol. Cell. Biol. 12, 1490±1499. Dutta, A., Stoeckle, M. Y., and Hanafusa, H. (1990). Serum and v-src increase the level of a CCAAT-binding factor required for transcription from a retroviral long terminal repeat. Genes Dev. 4, 243±254. Faber, M., and Sealy, L. (1990). Rous sarcoma virus enhancer factor 1 is a ubiquitous CCAAT transcription factor highly related to CBF and NF-Y. J. Biol Chem. 265, 22234±22254. Garnier, J. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97±120. Gelman, I. H., and Hanafusa, H. (1993). src-specific immune regression of Rous sarcoma virus-induced tumors. Cancer Res. 53, 915±920. Gonneville, L., Martins, T. J., and BeÂdard, P-A. (1991). Complex expression pattern of the CEF-4 cytokine in transformed and mitogenically stimulated cells. Oncogene 6, 1825±1833. Grewal, I. S., Rutledge, B. J., Fiorillo, J. A., Gu, L., Gladue, R. P., Flavell, R. A., and Rollins, B. J. (1997). Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1. J. Immunol. 159, 401±408. Heinemeyer, T., Wingender, E., Reuter, I., Hermjakob, H., Kel, A. E., Kel, O. V., Ignatieva, E. V., Ananko, E. A., Podkolodnaya, O. A., Kolpakov, F. A., Podkolodny, N. L., and Kolchanov, N. A. (1998). Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res. 26, 362±367. Lenardo, M. J., Fan, C.-M., Maniatis, T., and Baltimore, D. (1989). The involvement of NF-B in -interferon gene regulation reveals its role as widely inducible mediator of signal transduction. Cell 57, 287±294. Li, Q.-J., and Martins-Green, M. (1998). Stimulation of 9E3/ cCAF chemokine gene expression by thrombin occurs via the Map kinase cascade. Mol. Biol. Cell Suppl. 9, 231a. Li, Q.-J, Sladek, F., and Martins-Green, M. (1998). Thrombin stimulates transcription of the 9E3/cCAF chemokine gene through Elk-1 binding elements. Mol. Biol. Cell Suppl. 9, 194a. Li, Q.-J., Vaingankar, S., Green, H. M., and Martins-Green, M. (1999). Activation of the 9E3/cCAF chemokine by phorbol esters occurs via multiple signal transduction pathways that converge in MEK1/ERK2 and activate the Elk1 transcription factor. J. Biol. Chem. 274, 15454±15465. Lubkowski, J., Bujacz, G., Boque, L., Domaille, P. J., Handel, T. M., and Wlodawer, A. (1997). The structure of MCP-1 in two crystal forms provides a rare example of variable quaternary interactions. Nature Struct. Biol. 4, 64±69. Mackie, E. J., Halfter, W., and Liverani, D. (1988). Induction of tenascin in healing wounds. J. Cell Biol. 107, 2757±2767.

9E3/cCAF 1141 Martins-Green, M. (1993). Gro genes and wound repair: Overexpression of 9E3/CEF4 and its relationship to cell growth and wound healing. J. Cell. Biochem. Suppl. 17E, 105. Martins-Green, M., and Bissell, M. J. (1990). Localization of 9E3/ CEF-4 in avian tissues: expression is absent in Rous sarcoma virus-induced tumors but is stimulated by injury. J. Cell Biol. 110, 581±595. Martins-Green, M., and Feugate, J. E. (1998). The 9E3/CEF4 gene product is a chemotactic and angiogenic factor that can initiate the wound healing cascade in vivo. Cytokine 10, 522±535. Martins-Green, M., and Hanafusa, H. (1997). The 9E3/CEF4 gene and its product the chicken chemotactic and angiogenic factor (cCAF): potential roles in wound healing and tumor development. Cytokine Growth Factor Rev. 8, 221±232. Martins-Green, M., and T. Kelly (1998). The chicken chemotactic and angiogenic factor (cCAF): its angiogenic properties reside in the C-terminus of the molecule. Cytokine 10, 819±829. Martins-Green, M., and Li, Q.-J. (1999). Stimulation of cCAF by thrombin occurs via the MAP kinases MEK1/ERK2 with subsequent activation of the ELK1 transcription factor. Keystone Symposium on Chemokines and their Receptors, 35, 117a. Martins-Green, M., Tilley, C., Schwarz, R., Hatier, C., and Bissell, M. J. (1991). Wound-factor-induced and cell cycle phase-dependent expression of 9E3/CEF4, the avian gro gene. Cell Regul. 2, 739±752. Martins-Green, M., Aotaki-Keen, A., Hjelmeland, L., and Bissell, M. J. (1992). The 9E3 protein: immuno-localization in vivo and evidence for multiple forms in culture. J. Cell Science 101, 701±707. Martins-Green, M., Stoeckle, M., Hampe, A., Wimberly, S., and Hanafusa, H. (1993). Biochemical characterization of the 9E3/ CEF4 protein and its interaction with extracellular matrix. Mol. Biol. Cell Suppl. 4, 1742a. Martins-Green, M., Stoeckle, M., Wimberly, S., Hampe, A., and Hanafusa, H. (1996). The 9E3/CEF4 cytokine: kinetics of secretion, processing by plasmin, and interaction with extracellular matrix. Cytokine 8, 448±459. Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Apella, E., Kung, H. F., Leonard, E. J., and Oppenheim, J. J. (1988). Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor. J. Exp Med. 167, 1883±1893. Morimoto, R. I. (1993). Cells in stress: transcriptional activation of heat shock genes. Science 259, 1409±1410. Oquendo, P., Alberta, J., Wen, D., Graycar, J. L., Derywek, R., and Stiles, C. D. (1989). The platelet-derived growth factorinducible KC gene encodes a secretory protein related to platelet -granule proteins. J. Biol Chem. 264, 4133±4137. Petrenko, O., Ischenko, I., and Enrietto, P. J. (1995). Isolation of a cDNA encoding a novel chicken chemokine homologous to mammalian macrophage inflammatory protein-1 beta. Gene 160, 305±306. Qureshi, S. A., Joseph, C. K., Rim, M., Maroney, A., and Foster, D. A. (1991). v-src activates both protein kinase Cdependent and independent signaling pathways in murine fibroblasts. Oncogene 6, 995±999. Richmond, A., Lawson, D. H., Nixon, D. W., Stevens, J. S., and Chawla, R. K. (1982). In vitro growth promotion in human

malignant melanoma cells by fibroblast growth factor. Cancer Res. 42, 3175±3180. Richmond, A., Lawson, D. H., Dixon, D. W., Stevens, J. S., Chawla, R. K. (1983). Extraction of a melanoma growthstimulatory activity from culture medium conditioned by the Hs0294 human melanoma cell line. Cancer Res. 43, 2106±2112. Schmitt, M., Wilhelm, O., Janicke, F., Magdolen, V., Reuning, U., Ohi, H., Moniwa, N., Kobayashi, H., Weidle, U., and Graeff, H. (1995). Urokinase-type plasminogen activator (uPA) and its receptor (CD87): a new target in tumor invasion and metastasis. J. Obstet. Gynecol. 21, 151±165. Sick, C., and Staeheli, P. (1997). Cloning of a novel C-X-C chemokine secreted by LPS-induced chicken macrophages. J. Interferon Cytokine Res. 17, S96. Spangler, R., Joseph, C., Qureshi, S. A., Berg, K. L., and Foster, D. A. (1989). Evidence that v-src and v-fps gene products use a protein kinase C-mediated pathway to induce expression of a transformation-related gene. Proc. Natl Acad. Sci. USA 86, 7017±7021. Stoeckle, M. Y., and Barker, K. A. (1990). Two burgeoning families of platelet factor 4-related proteins: mediators of the inflammatory response. New Biologist 2, 313±323. Stoeckle, M. Y., and Hanafusa, H. (1989). Processing of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virus-transformed cells. Mol. Cell. Biol. 9, 4738±4745. Sugano, S., Stoeckle, M. Y., and Hanafusa, H. (1987). Transformation by Rous sarcoma virus induces a novel gene with homology to a mitogenic platelet protein. Cell 49, 321±328. Torre, E. A., and Fulco, R. A. (1996). Tumor-associated urokinase-type plasminogen activator: significance in breast cancer. Eur. J. Gynaecol. Oncol. 17, 315±318. Vaingankar, S., and Martins-Green, M. (1998). Thrombin activation of the 9E3/CEF4 chemokine involves tyrosine kinases including C-Src and the EGF receptor. J. Biol. Chem. 273, 5226±5234. Walz, A., Peveri, P., Aschauer, H., and Bagglioni, M. (1987). Purification and amino acid sequencing of NAF, a novel neutrophil-activating factor produced by monocytes. Biochem. Biophys. Res. Commun. 149, 755±761. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Appella, E., Oppenheim, J. J., and Leonard, E. J. (1987). Purification of human monocyte-derived neutrophil chemotactic factor that has peptide sequence similarity to other host defense cytokines. Proc. Natl Acad. Sci. USA 84, 9233±9237.

ACKNOWLEDGEMENTS I am in debt to my young postdoctoral and graduate student colleagues as well as to the technicians and undergraduates who have worked on this project over the years. I also thank ACG Design for help in preparing Figures 2 and 5 of this chapter. This work was supported by NIH grant no. GM48436, University of California Cancer Research Coordinating Committee grant, and the University of California, Riverside.