The present evaluates the key features of the WFDC1 [WAP (whey acidic protein) four disulfide core 1] gene that encodes ps20 (20 kDa prostate stromal protein), a member of the WAP family. ps20 was first characterized as a growth inhibitory activity that was secreted by fetal urogenital sinus mesenchymal cells. Purified ps20 exhibited several activities that centre on cell adhesion, migration and proliferation. The WFDC1 gene was cloned, contained seven exons, and was mapped to chromosome 16q24, suggesting that it may function as a tumour suppressor; however, direct evidence of this has not emerged. In vivo, ps20 stimulated angiogenesis, although expression of WFDC1/ps20 was down-regulated in the reactive stroma tumour microenvironment in prostate cancer. WFDC1 expression is differential in other cancers and inflammatory conditions. Recent studies point to a role in viral infectivity. Although mechanisms of action are not fully understood, WFDC1/ps20 is emerging as a secreted matricellular protein that probably affects response to micro-organisms and tissue repair homoeostasis.
WAPs (whey acidic proteins) and tissue homoeostasis
The WAP family has evolved to exhibit core functions that involve antimicrobial, immune and tissue homoeostasis activities. Each are secreted proteins with at least one WFDC (WAP four-disulfide core) domain that contains eight cysteine residues, which form four disulfide bonds in the mature form. The WAP and the WFDC domain were named by Hennighausen and Sippel in 1982  in a study of murine milk proteins. Most WAP family members exhibit protease inhibitor activity and, where examined, antimicrobial activity. Although the full-length proteins show differential conservation, the WFDC domain is reasonably well conserved. WAP proteins are secreted from a variety of cell types and have a wide variety of functions, including cell adhesion, migration and proliferation. Although activities are diverse, as a class of proteins, the WAP proteins generally function in some type of host response to tissue wounding and/or microbial infections. Moreover, they exhibit functions in immediate response not only to altered tissue homoeostasis but also to subsequent repair processes. These manifest as alterations in cell recruitment, migration and ECM (extracellular matrix) remodelling. Furthermore, some WAP members affect tissue repair-associated events such as angiogenesis. Most of these activities function to restore adult tissue to homoeostasis after wounding and infection. Accordingly, it is tempting to place the WAP proteins into a generalized functional category of proteins that respond to disrupted tissue homoeostasis through innate antimicrobial activity and subsequent repair responses. It should be noted, however, that activities of the WAP family of proteins are diverse and in many cases remain poorly understood. Certainly, this is an understudied field.
The biological actions of UGIF (urogenital sinus-derived growth inhibitory factor)/ps20 (20 kDa prostate stromal protein)
Our group began studying the biological activity of ps20, the protein encoded by WFDC1, in the mid 1980s. The first publication was based on our interest in interactions between mesenchymal and epithelial cells in the development of the urogenital sinus to the prostate gland. Fetal rat urogenital sinus-derived mesenchymal cells were isolated and an interesting biological activity that affected epithelial cell proliferation and protein synthesis was noted. This activity was termed UGIF . Of interest, cancer cells, including the NBT (Nara Bladder Tumour)-II cells, were particularly responsive to UGIF activity. We noted that mesenchymal cells and adult prostate stromal cells secreted relatively high levels of UGIF activity when cultures reached confluence and formed foci of spheroids. The degree of UGIF activity correlated temporally with the formation of spheroid foci in extended confluent cultures . Hence, UGIF activity correlated with cessation of proliferation and formation of three-dimensional spheroids in confluent cultures.
Purification of ps20
The next step in evaluating UGIF activity was to purify the protein responsible, ps20. Serum-free conditioned medium was collected from spheroids that were maintained in culture for 3–6 months. Curiously, the high expression of ps20 in three-dimensional spheroids is not well understood, yet it was important for generating enough protein to purify. The combination of ammonium sulfate precipitation, ion-exchange chromatography, gel-filtration chromatography and reverse-phase HPLC was used to purify ps20 to homogeneity . We found that ps20 co-purified with TGFβ1 (transforming growth factor β1) and β2 microglobulin and that β2 microglobulin stimulated proliferation and antagonized the well-documented growth-inhibitory activity of TGFβ . ps20 exhibited a molecular mass of 20–21 kDa, was growth inhibitory and stimulated protein synthesis in prostate cancer cells . Under reducing (unfolded) conditions, ps20 was 27–29, and 21–23 kDa under non-reducing conditions. Amino acids 1–14 and 19–28 were determined and verified that ps20 was a novel protein. We theorize that spheroids were initiated at focal sites of mesenchymal stem cells activated to expand in three-dimensional structures and that ps20 was a component of the inner core of ECM proteins in these spheroids. When considered in view of what we have learned subsequently, these findings fit with the hypothesis that ps20 may function as a matricellular protein that regulates stem cell niches and adult differentiated cell biology.
Structure of the
WFDC1 gene that encodes ps20
On the basis of the available amino acid sequence, degenerate primers were made, and PCR was used to isolate N-terminal fragments of the rat cDNA encoding ps20. These fragments were used for screening to isolate full-length (1029 bp) cDNA clones. The cDNA encoded a 212-amino acid protein containing a hydrophobic leader sequence and signal peptidase cleavage site immediately before a 186 amino acid mature form (20.7 kDa predicted size) . The only identifiable domain was the 44-amino acid WFDC domain (amino acids 57–101). An antibody was generated and we showed that ps20 was expressed in stromal cells in the native prostate gland. Interestingly, immunoreactivity was particularly evident in smooth muscle and stromal cells in vessel walls (Figures 1A and 1B). Overexpression of the cDNA in COS-7 cells resulted in a reduced proliferation, abnormal morphology and piling of the cells, suggesting that recombinant ps20 activity was affecting cell adhesion, migration and a piling-spheroid formation in a manner consistent with observations using the native purified protein.
Immunohistochemistry of WFDC1/ps20 expression in human prostate gland
To understand the structure of WFDC1, we next cloned the human cDNA and the mouse and human genomic sequences . These studies showed that both the mouse and human WFDC1 gene were composed of seven exons with a 79.1% identity. The WFDC domain sequence was contained in exon 2 and was well conserved, being nearly identical between mouse and human and was the region of highest amino acid homology between rat, mouse and human. Overall, the mouse and human mature ps20 share an 87.7% amino acid sequence identity. Interestingly, we also showed that the human WFDC1 gene mapped to chromosome 16q24, a telomere region that exhibits frequent LOH (loss of heterozygosity) in many cancers. Given the known growth inhibitor activity of ps20, this suggested that WFDC1 might function as a tumour-suppressor gene.
WFDC1 and tissue homoeostasis
Several studies have addressed the function of WFDC1/ps20 in tissue homoeostasis. Initially, the concept that WFDC1 may function as a tumour-suppressor gene seemed possible, given the known growth inhibitory properties of ps20 . This was further substantiated by Watson et al. , who used comparative genomic hybridization to show that considerable genomic loss occurred between 16q23.1 and 16qter in prostate cancer tissue samples. Concurrently, we showed that decreased ps20 expression was associated with reactive stroma in human prostate cancer and that this decrease was predictive of biochemical recurrence [elevation of PSA (prostate-specific antigen)] in prostate cancer patients after radical prostatectomy . Watson et al.  went on to show that expression of WFDC1 correlated with the amount of stroma, and that it was differentially expressed in prostate cancer, where no mutations were observed. However, a novel splice form, where exon 3 was deleted, was detected in both prostate cancer and normal prostate tissue. Similar to our findings, these studies showed that expression of WFDC1 was down-regulated in prostate cancer . Together, these studies show that WFDC1 expression is changed in prostate cancer progression but that the gene is not frequently mutated, indicating that WFDC1 probably does not function as a classical tumour-suppressor gene. Additional studies have provided little evidence to support the notion that WFDC1 is a tumour-suppressor gene. Saffroy et al.  showed that WFDC1 was unlikely to play a role in hepatocellular cancer although LOH in the WFDC1 locus was detected. Similarly, in breast cancer, WFDC1 shows infrequent mutations in stromal cells  and WFDC1 expression is not affected in retinoblastoma, where 16q24 is a commonly altered (LOH) locus .
The down-regulation of WFDC1/ps20 in prostate cancer was the first indication that the tumour microenvironment stroma was different than normal stroma (Figures 1C–1E). Owing to these clues, we went on to later define other markers including decreased calponin, elevated tenascin-C, pro-collagen I and fibroblast activation protein in reactive stroma cells [14,15]. These studies, for the first time, defined reactive stroma phenotype in prostate cancer and were the impetus for designing and completing studies that showed the tumour promoting activity in reactive stroma [16–20].
Unlike most of the other WAP family genes, WFDC1 is not located on chromosome 20q13 where 14 other WAP family members and related genes are located . This suggests that WFDC1 evolved with a different ancestral origin. What is perhaps most notable about the chromosome 20 WAP gene cluster is that it is one of best examples of a region that is undergoing ‘adaptive evolution’ and ‘rapid diversification’ in primates . The functional significance of this is not well understood. Interestingly, unlike WFDC1, most of these other WAP family genes either exhibit no change in expression or exhibit elevated expression in cancer . For example, HE4 is a well-characterized marker of ovarian cancer [24,25], as is elafin . It is clear that WFDC1 is unique in its evolution as compared with many other WAP family genes. Although ps20 exhibits some biological activities similar to other WAP family members, it is likely that WFDC1/ps20 may exhibit distinct functions, not necessarily associated with other chromosome 20 WAP genes.
Others have studied WFDC1/ps20 in reactive stroma biology associated with epithelial cancers. Similar to our reported data, Madar et al.  found that WFDC1 expression was down-regulated in CAFs (carcinoma associated fibroblasts) and inhibited the proliferation of HT1080 fibrosarcoma cells. Interestingly, this group also found that WFDC1 expression was elevated in senescent fibroblasts and they suggested that decreased WFDC1 might function as a biomarker of cell transformation. This is fully consistent with data showing that decreases WFDC1/ps20 correlated with a more aggressive human prostate cancer.
It is becoming clearer that WFDC1/ps20 exhibits multiple complex functions with many cell types. For example, WFDC1/ps20 was shown to regulate angiogenesis in the tumour microenvironment . Using a recombined cell xenograft model, we showed that WFDC1 overexpressed in stromal cells resulted in elevated microvessel density and a net increase in rate of tumorigenesis , although our earlier studies showed that ps20 inhibited prostate cancer cells proliferation in culture [4,6]. These studies also showed that ps20 stimulated endothelial migration  and the expression of VCAM1 (vascular cell adhesion molecule 1) (S.J. McAlhany, S.J. Ressler and D.R. Rowley, unpublished work). WFDC1/ps20 was expressed in the vessel wall, primarily in smooth muscle. Although we reported that WFDC1/ps20 is down-regulated in reactive stroma, interestingly, patients with higher biopsy Gleason scores, also showed an increase in WFDC1/ps20 in cancer epithelial cells . Hence, WFDC1/ps20 function in cancer is likely to be complex, with activity affecting both cancer cells and the tumour microenvironment, including blood vessel biology. Owing to these differential activities, it is difficult to predict what the net effects of altered WFDC1/ps20 expression might mean to the evolving biology of any particular tumour system. Overexpression in the stromal cells in a recombined prostate cancer xenograft tumour model system showed that elevated expression of WFDC1/ps20 in stroma resulted in a net tumour growth rate, concurrent with elevated angiogenesis . A role for WFDC1/ps20 in vascular biology is also supported by findings that WFDC1/ps20 is expressed at higher levels in the heart and lungs as compared with other tissues [6, 28].
Less clear are the mechanisms that regulate WFDC1/ps20 expression. Interestingly, WFDC1/ps20 expression was negatively regulated by oestrogen in the rat uterus, with differential expression noted during uterine cycles . These studies also showed that ps20 expressed in MCF-7 cells inhibited PC3 prostate cancer cell proliferation, similar to our findings. These authors concluded that ps20 is a local regulator of uterine growth. It is quite likely that epigenetic mechanisms also regulate WFDC1 expression. Methylation of the WFDC1 promoter is observed in melanoma samples, although at a lower level as compared with several other genes . This same group showed that overexpression of WFDC1 inhibited the tumorigenesis of melanoma cells in vivo . In addition, these studies showed that Dkk1 (Dickkopf-1) was overexpressed in cells engineered to overexpress WFDC1. This suggested that the growth inhibitory function of WFDC1/ps20 may be the result of Dkk1 action, as an inhibitor of the Wnt signalling pathway. Also of interest, overexpression of latexin induced the WFDC1 gene in human gastric cancer cells and these cells were growth inhibited both in vitro and in vivo . Latexin, a carboxypeptidase inhibitor, functions to inhibit the proliferation of gastric cancer cells, along with mouse stem cells. Again, in these studies, WFDC1 expression negatively correlated with tumour growth. This seems to be a generally consistent finding.
It is also clear that the expression and biological activity of WFDC1/ps20 are associated with inflammatory conditions. In rheumatoid arthritis, WFDC1 is involved in ‘epistatic interactions’ with PTPN22, a susceptibility factor for rheumatoid arthritis . Given the recent findings by Alvarez et al. [33,34] that WFCD1 plays a role in susceptibility of T-cells to HIV infection, it is becoming even clearer that the WFDC1 gene is important to immune system functions and innate immunity and is likely to be involved in several complex biological processes .
There is additional evidence that WFDC1/ps20 functions in the nervous system and associated tissues. WFDC1 is expressed differentially in human RPE (retinal pigment epithelium) in the macular compared with the peripheral extramacular regions [36,37]. WFDC1 was one of the most highly differentially expressed genes and this was confirmed via immunohistochemistry. ECM genes were the most significant class of differentially expressed genes. This is of interest, because the only clear association of WFDC1 with a specific disease has been multiple eye defects in bovines that are associated with chromosome 18. These disorders were associated with an insert in the WFDC1 gene that produced a frame shift . Clearly, additional studies are required to dissect key mechanisms.
It also should be noted that structurally ps20 has several similarities with the Kallmann's syndrome protein, anosmin-1, encoded by the KAL1 gene. Anosmin-1 was shown to interact directly with FGFR1 (fibroblast growth factor receptor 1) [39,40] and the FNIII (fibronectin type III domain) repeats and the WFDC cysteine residues were critically important for this collective interaction . The integrated actions of anosmin-1, FGF-2 (fibroblast growth factor-2) and FGFR1 were important for chemoattraction and motogenic effects on neuronal progenitors during development of the rostral migratory stream of the forebrain–olfactory bulb  and outgrowth of Purkinje axons in cerebellum . To our knowledge, the direct interaction of ps20 with FGFR1 has yet to be studied, but this potential interaction could explain the pro-angiogenesis function of ps20, since FGFR signalling is a critical component of angiogenesis during tissue repair and remodelling processes.
Interestingly, expression of KAL1 by keratinocytes was up-regulated by TGFβ1 , similar to what we reported for WFDC1/ps20 in prostate stromal cells . In addition, anosmin-1 exhibits decreased expression in early stages of colon, lung and ovarian cancer, and an increase in later stages . This expression was regulated by both hypoxia and TGFβ and anosmin-1 functioned to increase cancer cell motility and protected cancer cells from apoptosis. Again, several of these expression patterns and features are shared with what we currently know about WFDC1/ps20. Finally, anosmin-1 differentially affected adhesion of oligodendrocytes to ECM components, important for migration . We reported previously that WFDC1/ps20 affects cell adhesion and spheroid formation [3,4,6] and Watson et al.  suggested that ps20 may play a role in maintaining ECM in the stroma. Taken together, these data suggest that several shared common biological functions have been reported between anosmin-1 and WFDC1/ps20 and that at least some of this biology is known to be attributed to the structure and function of the WAP domain in these proteins .
The first studies of WFDC1/ps20 were initiated as attempts to identify an unknown biological activity secreted by mesenchymal-fibroblast cells. Over two decades later, we now understand more about the structure and function of the WFDC1 gene and ps20, although much remains unknown. Clearly, WFDC1 expression leads to secretion of ps20. Splice variants exist, yet little is understood about the putative proteins encoded by these. It is likely that ps20 is a matricellular protein that affects cell adhesion, migration and proliferation. Binding partners are not well understood; however, it is clear that ps20 affects infectivity of cells to HIV and probably to other viruses. Like the other WAP family members, ps20 may have antimicrobial activity although this also is poorly understood. Expression of WFDC1/ps20 in general is decreased in the tumour microenvironment and has differential effects on cancer progression. Although ps20 probably exhibits growth inhibitory activity, it also stimulates angiogenesis in the microenvironment. Accordingly, the net effects of altered WFDC1/ps20 expression in any particular site of altered tissue homoeostasis are difficult to predict. It is likely that ps20 interacts with several proteins and is involved in different signalling pathways. Dissecting key mechanisms is necessary to fully understand the roles of WFDC1/ps20 in normal and diseased human tissues.
Structure and Function of Whey Acidic Protein (WAP) Four-Disulfide Core (WFDC) Proteins: An Independent Meeting held at Robinson College, Cambridge, U.K., 12–14 April 2011. Organized and Edited by Colin Bingle (Sheffield, U.K.), Judith Hall (Newcastle, U.K.), Cliff Taggart (Queen's University Belfast, U.K.) and Annapurna Vyakarnam (King's College London, U.K.).
This work was supported in part by the National Institutes of Health [grant numbers RO1 CA58093 and U54 CA126568].