Maturation of PDGFs

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PDGFs are a pleotrophic family of growth factors that stimulate various cellular functions including growth, proliferation, and differentiation. The expression of PDGFs has been demonstrated in a number of different solid tumors, from glioblas-tomas to colon carcinomas [10-13]. In these various tumor types, the biologic role of PDGF signaling can vary from autocrine stimulation of cancer cell growth to paracrine interactions involving adjacent stroma and vasculature leading to tumor progression, angiogenesis and metastasis [10-13].

PDGF are a disulfide-linked dimers composed of two polypeptide chains, denoted A and B and represented in vivo by the three PDGFs: PDGF-AA, PDGF-AB and

Figure 1. Processing of A- and B-chains of PDGF and activation of their corresponding receptors. The A and B chains are produced as precursor molecules (i) that form disulfide-bonded dimers, undergo proteolytic processing (ii) and induce dimerization of alpha and/or beta receptors (iii)

PDGF-BB [14, 15] that mediate their signals through activation of two tyrosine kinase receptors, PDGFR-a and -P (Figure 1).

Recently two new members of the PDGF family, PDGF-C and PDGF-D were reported. PDGF-CC act as a ligand for PDGFR-a [l6] and PDGF-DD as a ligand for PDGFR-P [17]. Of the PDGFs proven to be activated by the PC are PDGF-A [1] and PDGF-B [18]. PDGF-C and PDGF-D were found also to possess potential PC cleavage site [4], however, their processing by the PCs is not yet proven experimentally.

3.1.1 ProPDGF-A processing in the malignant phenotype of tumor cells

Following dimerization of PDGF-A monomers in the ER into a ~50 kDa form, this complex transits through the Golgi apparatus towards the trans Golgi Network (TGN) where it is proteolytically cleaved by the PCs and secreted as ~30 kDa dimeric product [1]. Although the Furin is the major convertase that process significantly pro-PDGF-A [1], under steady-state conditions, the convertases PC5, PACE4 and PC7 were also found able to mediate pro-PDGF-A processing [1]. Like various PC substrates, PDGF-A processing is inhibited by the naturally occurring PC inhibitors PC-prosegments of Furin (ppFurin), ppPC5, ppPACE4, and by the general PC inhibitors: the Furin-motif variants of a2-macroglobulin (a2-MGF) and serpina1-antitrypsin (a1-PDX) [1].

Like the other PDGFs, the binding of PDGF-A ligand to its receptor results in the autophosphorylation of the latter [1]. In turn, the PDGF receptor activates an enzyme cascade that includes various phosphorylating enzymes including PKC,

Ras, Raf, and MAPK [1]. Comparative analysis between wild type and mutant (at its PC site) PDGF-A revealed that the unprocessed PDGF-A lost its ability to mediate tyrosine phosphorylation of PDGF-A receptor and were unable to stimulate [3H]thymidine incorporation in vitro. In vivo experiments revealed that the injection of tumor cells expressing the unprocessed PDGF-A inhibited tumor growth progression [1]. This in vivo tumor growth inhibition by the mutated PDGF-A, could be explained by the potential action of the unprocessed proPDGF-A as a dominant negative [19]. Indeed, like the other PDGF ligands, interaction of PDGF-A with their receptors induces the dimerization of the receptor subunits and the formation of PDGFaR-PDGFaR homodimers [1]. The absence of fully processed PDGF-A may affect the dimerization of the corresponding receptors leading to a loss of biological activity. Also, the possible antagonist role of the PDGF-A mutant that may compete with the active PDGF-A for the PDGF receptors it's not ruled out.

3.1.2 Proprotein convertases in ProPDGF-B processing and secretion

Contrary to PDGF-A, ProPDGF-B is processed into a major cell surface-associated product of 24 kD and minor secreted product of 30 kD [18]. The ProPDGF-B newly synthesized as 31 kD monomers is rapidly dimerized in the ER via disulfide bridge bonds to yield a 56-60 kD proPDGF-B. The latter is transferred thereafter to the Golgi complex for proteolytic processing to produce an intermediate 40 kD and a final 27 kD product [18]. These forms of PDGF-B are the results of the processing action of Furin, PACE4, PC5, or PC7 that occurs in the N termini at pairs of basic residues RGRR81, followed by a second cleavage by unknown enzyme at the C termini at the surrounding amino acid sequence ARPVT19 [18]. This stepwise processing of PDGF-B is controlled by the processing at the PCs site RGRR81 as revealed by expression experiments with proPDGF-B mutated at its PC-cleavage site [RGRR81 into AGRA81). This mutation eliminated completely the processing of PDGF-B at both the N terminal and C terminal sites. The use of wild-types and mutants PDGF-A and/or PDGF-B chains cDNAs, revealed that the formation of all mature PDGFs isoforms, PDGF-AA, PDGF-BB and PDGF-AB require the processing of both the A and B chains [18]. This processing is also inhibited by a1-PDX and a2-MG-F and some endogenous PC inhibitors [18]. In contrast, the introduction at the second cleavage site of PDGF-B various mutations surrounding the amino acid sequence ARPVT190 that contain amino acid sequences bearing a resemblance to potential PC sites failed to block proPDGF-B processing.

The cleavage of PDGF-B at its second site seems to play an important role in the secretion of mature PDGF-B. Indeed, contrary to PDGF-A, the majority of the newly produced PDGF-B is retained at the cell surface, due to the presence of basic residues localized at the C-terminal end of the precursor within the segment aa 212-226. This PDGF-B cell retention motif is localized just after the second cleavage site of PDGF-B (ARPVT190). Mutation introduced at these areas reduced mature PDGF-B secretion, whereas overexpression of Furin resulted in an enhancement of the level of secreted PDGF-B [18]. Thus the processing of PDGF-B at its second site facilitates its secretion thereby the cleaved PDGF-B becomes more diffusible and may act on cells at some distance from the producer cell (Figure 2).

The attachment of the PDGF-B at the cell surface mediated by the residues 212-226 aa of the C-terminal end is due to its interaction with various components of the extracellular matrix, including heparan sulfate, thought to be its major interactor [20, 21]. This would result in the retention of the unprocessed PDGF-B at the cell surface, to be released in a controllable manner by a second processing event. Conversely, several studies reported that deletion of the retention motif in PDGF-B leads to its increased secretion and accumulation in the cell culture media [20, 21]. In addition, the reduced secretion of PDGF-B was previously reported to

Figure 2. PDGF-B processing and secretion. The newly produced PDGF-b is retained at the cell surface, due to the presence of basic residues localized at the c-terminal (CT) end of the precursor. This PDGF-B cell retention motif is localized just after the second cleavage site of PDGF-B (ARPVT190). The processing of PDGF-B at this second site facilitates its secretion thereby the cleaved PDGF-B becomes more diffusible and may act on cells at some distance from the producer cell

Figure 2. PDGF-B processing and secretion. The newly produced PDGF-b is retained at the cell surface, due to the presence of basic residues localized at the c-terminal (CT) end of the precursor. This PDGF-B cell retention motif is localized just after the second cleavage site of PDGF-B (ARPVT190). The processing of PDGF-B at this second site facilitates its secretion thereby the cleaved PDGF-B becomes more diffusible and may act on cells at some distance from the producer cell limit the action range of PDGF-B in vivo, as suggested from experiments with transplanted keratinocytes expressing wild type or retention motif-truncated PDGF-B into athymic mice [22].

3.2 Maturation of VEGF-C by the Proprotein Convertases in Tumor Angiogenesis

VEGF-C is secreted as a disulfide-bonded homodimer that is processed from the precursor polypeptide at the PC cleavage site HSIIRR227SL (Figure 3). This processing is mediated mainly by the convertases Furin, PC5, and PC7 dividing it into N-terminal (31 kDa) and cysteine-rich C-terminal (29 kDa) polypeptides [2]. Cellular coexpression and in vitro kinetics analysis revealed that Furin and PC5 are the most effective proVEGF-C convertases [2]. After Pro-VEGF-C cleavage by the PC an additional processing removes the N-terminal propeptide and generates the 21-kDa VEGF-C (Figure 3).

Figure 3. Schematic representation of the primary structure of the 419-AA human proVEGF-C and its receptors R2 and R3. Shown are the signal peptide (SP), PC-processing site (HSIIRR227 SL), an unknown protease site (indicated by question mark) that generates the 21-kDA VEGF-C. Activation of VEGFR-2 induces blood vessel formation and activation of VEGFR-3 induces lymphatic vessels formations

Figure 3. Schematic representation of the primary structure of the 419-AA human proVEGF-C and its receptors R2 and R3. Shown are the signal peptide (SP), PC-processing site (HSIIRR227 SL), an unknown protease site (indicated by question mark) that generates the 21-kDA VEGF-C. Activation of VEGFR-2 induces blood vessel formation and activation of VEGFR-3 induces lymphatic vessels formations

The protease (s) that mediates this conversion occurring extracellularly and/or at the cell surface is still unknown. The intracellular proteolytic cleavage of pro-VEGF-C is not a prerequisite for VEGF-C secretion as revealed by the accumulation of proVEGF-C in media derived from the Furin deficient cells LoVo transfected with VEGF-C and following the expression of PC inhibitors in various cells [2]. Of the PC inhibitors that were found to inhibit the processing of VEGF-C the prosegments, ppFurin, ppPC5, ppPACE4, and the serpins a1-PDX, and a2-MG-F [2]. VEGF-C overexpression was linked to various cancers and metastasizing tumors in which VEGF-C was shown to mediate vessel formation [1, 23-25].

Although, it was originally described as a specific growth factor for lymphatic vessels and a ligand for the lymphatic endothelial receptor VEGFR-3 (Flt4), VEGF-C was found also to bind and activates VEGFR-2, which is the major mitogenic signal transducer for VEGF in blood vessel endothelial cells [23-25] (Figure 3). Recently, expression of VEGF-C in CHO cells, failed to stimulate tumor growth in vitro and in vivo [2], but stimulated both tumor lymphangiogenesis and angiogenesis [2].

The inability of VEGF-C to stimulate cell proliferation is probably due to its weak mitogenic action as compared with the other VEGFs. Indeed, VEGF-C is 50-to 100-fold less potent than VEGF in inducing proliferation of endothelial cells [23-25]. In contrast, VEGF-C is a potent angiogenic factor, and acts synergistically with other growth factors to mediate angiogenesis [25]. Blockade of VEGF-C processing by mutagenesis generates predominately the 59-kDa proVEGF-C form [2]. This form was reported to behave as an antagonist of VEGFR-2 and VEGFR-3 [1, 21-25], and to possibly prevent their optimal oligomerization, which is needed for their signaling functions. Accordingly, the observed reduction in tumor growth, angiogenesis, and lymphangiogenesis of the 59-kDa proVEGF-C-producing tumors

[2] suggested to be related to the dominant-negative characteristics of this growth factor [2]. Recently, an opposing role of unprocessed and processed PC substrates was reported for other proteins and demonstrated to be related to distinct signaling pathways. This includes pro-nerve growth factor (proNGF) and other neurotrophins

[3], suggesting that similar mechanism may explain the divergent action of the unprocessed and processed pro-VEGF-C on tumorigenesis.

3.3 Maturation of IGFs by the Proprotein Convertases

Although the IGF-1 isolated from human serum is 70 amino acids long, after its production as a precursor protein, the processing of pro-IGF-I to mature IGF-I occurs by cleavage within the processing motif PLKPAKSAR71 ; RSVRAQR77 ; HT at two sites : Arg71 to generate IGF-I-(1-70) and at Arg77 to produce IGF-I-(1-76) [26, 27]. The mature IGF-1 form is generated from the two different protein precursors; pro-IGF-IA and pro-IGF-IB.

These molecules are the products of a single gene by alternative splicing that circulate in human serum as a peptide consisting of four domains namely B, C, A and D [26-28]. The sequences of IGF-IA and IGF-IB are identical through B, C, A, and D and through the 15 first amino acid of the pro-region or E domain and both possess the same processing site between E and D domains [26-28]. Previously it was reported that when pro-IGF-1 was expressed in RPE.40 cells, a CHO-K1 derivate cells lacking furin activity, the cleavage of pro-IGF-1 occurs at Arg71 but not at Arg77. Similar results were obtained when pro-IGF-1 was expressed in the furin deficient colon cancer cells LoVo, suggesting that furin is required for cleavage at Arg77 but not at Arg71 [29]. Later, Duguay SJ et al., by expressing wild type and mutant forms of Pro-IGF-1 revealed the preference of Furin to Arg77 and the processing of proIGF-1 at Arg71 by the other members of the proprotein convertases [26, 27].

Similarly, The forms of IGF-2 that are most abundant in normal serum are the mature IGF-2 (1-67) and IGF-2-(1-87) [30]. Expression experiments with pro-IGF-2 mutants containing substitutions at potential cleavage sites revealed that pro-IGF-2 is processed at Arg104 in HK 293 cells [30]. Coexpression of pro-IGF-2 with furin, PACE4, PC5A, PC5B, or PC7 resulted in enhanced or complete processing of pro-IGF-2 confirming the cleavage of IGF-2 by the PCs. Contrary to IGF-1 of witch the processing occurs only intracellularly, proIGF-2 is processed during transport through the cell and extracellularly [30].

In addition to its critical role in the growth and development of many tissues and the regulation of the overall growth, particularly prenatal one, IGF-1 is also implicated in various pathophysiological conditions, and is thought to play a particularly prominent role in tumorigenesis. Previously, the potent mitogenic activity of IGF-I in cell culture made it an obvious candidate risk factor in cancer development. To date, a significant amount of data had been accumulated suggesting the important role of this growth factor in various cancers including prostate, breast, colon and lung cancers [31-36]. In the prostate, IGF-I is a mitogen for prostate epithelial cells. Various clinical studies revealed strong positive association between IGF-I levels and prostate cancer risk [31]. In breast cancer, also a positive relation between circulating IGF-I concentration and risk of breast cancer was found among premenopausal but not postmenopausal women, suggesting the use of plasma IGF-I concentrations in the identification of women at high risk of breast cancer [32]. Similarly, in colorectal [33] and lung [34] cancers various studies have reported positive associations between serum IGF-I and these cancer risks. Although investigators have compared the activity of mature and unprocessed IGFs in several in vitro assays such as thymidine incorporation, to date the importance of IGF-1 and IGF-2 processing by the PCs in the malignant phenotype of tumor cells and tumor progression is still not yet proven experimentally.

3.4 PTH and PTH-rP Processing by the PCs and Tumorigenesis

Both PTH and PTHrP hormones are initially synthesized as larger inactive precursor proteins that undergo processing to release the active molecules [37-42]. While PTH is involved in the regulation of normal extracellular fluid calcium homeostasis and expressed almost exclusively in the parathyroid gland, pro-PTHrP is widely expressed in both none-endocrine and neuroendocrine cells and considered as the major pathogenic endocrine factor in malignancy-associated hypercalcemia and bone metastasis [43]. Usually, primary cancers metastasize to bone by a multistep process that involves interactions between tumor cells and normal host cells. In breast carcinoma, the final step in bone metastasis is mediated by osteoclasts that are stimulated by local production of the tumor peptide PTH-rP. During this step PTHrp mediate bone destruction prior metastasis. In contrast in prostate carcinomas PTHrp stimulates osteoblasts to make new bone.

PTH mRNA, encodes a signal sequence of 25 amino acids and a basic pro-peptide of 6 amino acids [40-42] that constitute the proPTH. The latter is then transported to the trans-Golgi network where it's processed and the mature PTH polypeptide of 84 amino acids is packaged into secretory granules. This processing event occurs in the parathyroid chief cell, in the trans-Golgi network rather than in secretory granules, which is consistent with processing of proPTH by furin or a furin-like enzyme (PC7) rather than PC1 or PC2. These data are supported by the co-expression of proPTH with furin but not with PC1 and PC2 in the parathyroid cell.

In human, tree mature peptide isoforms derived from pro-PTHrp were previously reported, namely the 139, 141 and 173 aa. These peptides were proposed to be translated from three different mRNAs generated by alternative splicing [38]. These peptide isoformes differ only at their COOH-terminus. The intracellular conversion of pro-PTHrp to the mature products 139, 141 and 173 aa is mediated by furin at the motif Arg-Leu-Lys-Arg. Additional cleavage occurs at these matures peptides with undetermined enzymes to generate the 1-36 peptide able to interact with the receptor. The importance of PTHrP on the malignant phenotype was previously reported by Liu et al.. The authors demonstrated that alteration of PTHRP processing diminish the hypercalcemic endocrine actions of PTHRP and reduce autocrine/paracrine effects of PTHRP on tumor cell growth in vitro and in vivo as well [44].

3.5 Processing of Endothelin-1 by the Proprotein Convertases

By acting directly on endothelial cells, endothelin-1 (ET-1), was reported to modulate different stages of neovascularization, including proliferation, migration, invasion, proteases production and morphogenesis [45, 46]. ET-1 was found also able to modulate tumor angiogenesis indirectly through the induction of various angiogenic factors such as vascular endothelial growth factor (VEGF) [46]. In addition, tumor cells were reported to express ET-1 receptor, suggesting the potential participation of ET-1 in the tumorigenic and/or metastatic potential of several tumor cells [46].

The processing of proET-1 is a multi-step event that requires the participation of at least three proteases [47-48]. This three-step process consisting of an initial proteolytic cleavage of the pro-endothelin-1 precursor to big endothelin-1, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-1 peptide to endothelin-1 by endothelin-converting enzyme (ECE). The conversion of pro-ET-1 to big-ET-1 is mediated by furin or PC7 [47, 48]. Introduction of a point mutation into PC cleavage site of the pro-ET-1 prevent its processing at the Arg-Ser-Lys-Arg motif. Co-transfection with ECE-1 cDNA revealed that the cleavage at Arg52 is not essential for its processing by ECE-1, but cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1 [47, 48].

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