Protease-activated receptor-2 promotes osteogenesis in skeletal mesenchymal stem cells at the expense of adipogenesis: Involvement of interleukin-6.

Bone marrow mesenchymal stem cells (MSCs) give rise to osteoblasts and adipocytes, with an inverse relationship between the two. The MSCs from protease-activated receptor-2 knockout (PAR2 KO) mice have a reduced capacity to generate osteoblasts. Here we describe the observation that PAR2 KO osteoblastic cultures generate more adipocytes than wildtype (WT) cultures. Osteoblasts from PAR2 KO mice expressed lower levels of osteoblastic genes (Runx2, Col1a1 and Bglap), and higher levels of the adipocytic gene Pparg than WT osteoblasts. Bone marrow stromal cells from PAR2 KO mice generated fewer osteoblastic colonies (assessed by staining for alkaline phosphatase activity and mineral deposition) and more adipocytic (Oil Red-O positive) colonies than cultures from WT mice. Similarly, cultures of the bone marrow stromal cell line (Kusa 4b10) in which PAR2 was knocked down (F2rl1 KD), were less osteoblastic and more adipocytic than vector control cells. Putative regulators of PAR2-mediated osteogenesis and suppression of adipogenesis were identified in an RNA-sequencing (RNA-seq) investigation; these include C1qtnf3, Gpr35, Grem1, Snorc and Tcea3, which were more highly expressed, and Cnr1, Enpep, Hmgn5, Il6 and Ramp3 which were expressed at lower levels, in control than in F2rl1 KD cells. Interleukin-6 (IL-6) levels were higher in medium harvested from F2rl1 KD cells than from control cells, and a neutralising anti-IL-6 antibody reduced the number of adipocytes in F2rl1 KD cultures to that of control cultures. Thus, PAR2 appears to be a mediator of the reciprocal relationship between osteogenesis and adipogenesis, with IL-6 having a regulatory role in these PAR2-mediated effects.

MUC13 protects colorectal cancer cells from death by activating the NF-κB pathway and is a potential therapeutic target.

MUC13 is a transmembrane mucin glycoprotein that is over produced by many cancers, although its functions are not fully understood. Nuclear factor-κB (NF-κB) is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently upregulating BCL-XL. MUC13 promoted tumor necrosis factor (TNF)-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NF-κB essential modulator. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signaling in response to both TNF and DNA-damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to killing by cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers.

Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling.

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2 antagonism. Developing PAR2 ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2 homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR2 models based on ORL-1 and PAR1, site-directed mutagenesis of PAR2 was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR2 signaling. Effects of 28 PAR2 mutations were examined for PAR2-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR2 mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca2+ induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2 TM regions for receptor activation by synthetic PAR2 agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR2 was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2 physiology.

Elevated CDCP1 predicts poor patient outcome and mediates ovarian clear cell carcinoma by promoting tumor spheroid formation, cell migration and chemoresistance.

Hematogenous metastases are rarely present at diagnosis of ovarian clear cell carcinoma (OCC). Instead dissemination of these tumors is characteristically via direct extension of the primary tumor into nearby organs and the spread of exfoliated tumor cells throughout the peritoneum, initially via the peritoneal fluid, and later via ascites that accumulates as a result of disruption of the lymphatic system. The molecular mechanisms orchestrating these processes are uncertain. In particular, the signaling pathways used by malignant cells to survive the stresses of anchorage-free growth in peritoneal fluid and ascites, and to colonize remote sites, are poorly defined. We demonstrate that the transmembrane glycoprotein CUB-domain-containing protein 1 (CDCP1) has important and inhibitable roles in these processes. In vitro assays indicate that CDCP1 mediates formation and survival of OCC spheroids, as well as cell migration and chemoresistance. Disruption of CDCP1 via silencing and antibody-mediated inhibition markedly reduce the ability of TOV21G OCC cells to form intraperitoneal tumors and induce accumulation of ascites in mice. Mechanistically our data suggest that CDCP1 effects are mediated via a novel mechanism of protein kinase B (Akt) activation. Immunohistochemical analysis also suggested that CDCP1 is functionally important in OCC, with its expression elevated in 90% of 198 OCC tumors and increased CDCP1 expression correlating with poor patient disease-free and overall survival. This analysis also showed that CDCP1 is largely restricted to the surface of malignant cells where it is accessible to therapeutic antibodies. Importantly, antibody-mediated blockade of CDCP1 in vivo significantly increased the anti-tumor efficacy of carboplatin, the chemotherapy most commonly used to treat OCC. In summary, our data indicate that CDCP1 is important in the progression of OCC and that targeting pathways mediated by this protein may be useful for the management of OCC, potentially in combination with chemotherapies and agents targeting the Akt pathway.

EGF inhibits constitutive internalization and palmitoylation-dependent degradation of membrane-spanning procancer CDCP1 promoting its availability on the cell surface.

Many cancers are dependent on inappropriate activation of epidermal growth factor receptor (EGFR), and drugs targeting this receptor can improve patient survival, although benefits are generally short-lived. We reveal a novel mechanism linking EGFR and the membrane-spanning, cancer-promoting protein CDCP1 (CUB domain-containing protein 1). Under basal conditions, cell surface CDCP1 constitutively internalizes and undergoes palmitoylation-dependent degradation by a mechanism in which it is palmitoylated in at least one of its four cytoplasmic cysteines. This mechanism is functional in vivo as CDCP1 is elevated and palmitoylated in high-grade serous ovarian tumors. Interestingly, activation of the EGFR system with EGF inhibits proteasome-mediated, palmitoylation-dependent degradation of CDCP1, promoting recycling of CDCP1 to the cell surface where it is available to mediate its procancer effects. We also show that mechanisms inducing relocalization of CDCP1 to the cell surface, including disruption of its palmitoylation and EGF treatment, promote cell migration. Our data provide the first evidence that the EGFR system can function to increase the lifespan of a protein and also promote its recycling to the cell surface. This information may be useful for understanding mechanisms of resistance to EGFR therapies and assist in the design of treatments for EGFR-dependent cancers.

Stratum basale keratinocyte expression of the cell-surface glycoprotein CDCP1 during epidermogenesis and its role in keratinocyte migration.

BACKGROUND: Epidermogenesis and epidermal wound healing are tightly regulated processes during which keratinocytes must migrate, proliferate and differentiate. Cell-to-cell adhesion is crucial to the initiation and regulation of these processes. CUB-domain-containing protein (CDCP)1 is a transmembrane glycoprotein that is differentially tyrosine phosphorylated during changes in cell adhesion and survival signalling, and is expressed by keratinocytes in native human skin, as well as in primary cultures. OBJECTIVES: To investigate the expression of CDCP1 during epidermogenesis and its role in keratinocyte migration. METHODS: We examined both human skin tissue and an in vitro three-dimensional human skin equivalent model to examine the expression of CDCP1 during epidermogenesis. To examine the role of CDCP1 in keratinocyte migration we used a function-blocking anti-CDCP1 antibody and a real-time Transwell™ cell migration assay. RESULTS: Immunohistochemical analysis indicated that in native human skin CDCP1 is expressed in the stratum basale and stratum spinosum. In contrast, during epidermogenesis in a three-dimensional human skin equivalent model, CDCP1 was expressed only in the stratum basale, with localization restricted to the cell-cell membrane. No expression was detected in basal keratinocytes that were in contact with the basement membrane. Furthermore, an anti-CDCP1 function-blocking antibody was shown to disrupt keratinocyte chemotactic migration in vitro. CONCLUSIONS: These findings delineate the expression of CDCP1 in human epidermal keratinocytes during epidermogenesis and demonstrate that CDCP1 is involved in keratinocyte migration.

Blocking of CDCP1 cleavage in vivo prevents Akt-dependent survival and inhibits metastatic colonization through PARP1-mediated apoptosis of cancer cells.

The CUB domain-containing protein-1 (CDCP1) is a transmembrane molecule that has recently been implicated in cancer progression. In this study we have established a novel mechanism for initiation of CDCP1-mediated signaling in vivo and demonstrated that specific 135→70-kDa processing of cell-surface CDCP1 by extracellular serine proteases is a prerequisite for CDCP1-dependent survival of cancer cells during metastasis. The in vivo cleavage of CDCP1 triggers a survival program involving recruitment of Src and PKCδ, Src-mediated phosphorylation of cell-surface-retained 70-kDa CDCP1, activation of Akt and suppression of PARP1-induced apoptosis. We demonstrate in vivo that phosphorylated Src, PKCδ and Akt all constitute activated elements of a CDCP1-signaling axis during tissue colonization of tumor cells. Preventing in vivo cleavage of CDCP1 with unique anti-CDCP1 antibodies, serine protease inhibitors or genetic modulation of the cleavage site in the CDCP1 molecule completely abrogated survival signaling associated with the 70-kDa CDCP1, and induced PARP1 cleavage and PARP1-mediated apoptosis, ultimately resulting in substantial inhibition of tissue colonization by tumor cells. The lack of CDCP1 cleavage in the lung tissue of plasminogen-knockout mice along with a coordinated reduction in tumor cell survival in a lung retention model, and importantly rescue of both by in vivo supplied plasmin, indicated that plasmin is the crucial serine protease executing in vivo cleavage of cell-surface CDCP1 during early stages of lung colonization. Together, our findings indicate that in vivo blocking of CDCP1 cleavage upstream from CDCP1-induced pro-survival signaling provides a potential mechanism for therapeutic intervention into metastatic disease.

TTYH2, a human homologue of the Drosophila melanogaster gene tweety, is located on 17q24 and upregulated in renal cell carcinoma.

Using differential display PCR, we identified a novel gene upregulated in renal cell carcinoma. Characterization of the full-length cDNA and gene revealed that the encoded protein is a human homologue of the Drosophila melanogaster Tweety protein, and so we have termed the novel protein TTYH2. The orthologous mouse cDNA was also identified and the predicted mouse protein is 81% identical to the human protein. The encoded human TTYH2 protein is 534 amino acids and, like the other members of the tweety-related protein family, is a putative cell surface protein with five transmembrane regions. TTYH2 is located at 17q24; it is expressed most highly in brain and testis and at lower levels in heart, ovary, spleen, and peripheral blood leukocytes. Expression of this gene is upregulated in 13 of 16 (81%) renal cell carcinoma samples examined. In addition to a putative role in brain and testis, the over-expression of TTYH2 in renal cell carcinoma suggests that it may have an important role in kidney tumorigenesis.

Characterization of a novel gene, STAG1/PMEPA1, upregulated in renal cell carcinoma and other solid tumors.

Using differential display-polymerase chain reaction, we identified a novel gene sequence, designated solid tumor-associated gene 1 (STAG1), that is upregulated in renal cell carcinoma (RCC). The full-length cDNA (4839 bp) encompassed the recently reported androgen-regulated prostatic cDNA PMEPA1, and so we refer to this gene as STAG1/PMEPA1. Two STAG1/PMEPA1 mRNA transcripts of approximately 2.7 and 5 kb, with identical coding regions but variant 3' untranslated regions, were predominantly expressed in normal prostate tissue and at lower levels in the ovary. The expression of this gene was upregulated in 87% of RCC samples and also was upregulated in stomach and rectal adenocarcinomas. In contrast, STAG1/PMEPA1 expression was barely detectable in leukemia and lymphoma samples. Analysis of expressed sequence tag databases showed that STAG1/PMEPA1 also was expressed in pancreatic, endometrial, and prostatic adenocarcinomas. The STAG1/PMEPA1 cDNA encodes a 287-amino-acid protein containing a putative transmembrane domain and motifs that suggest that it may bind src homology 3- and tryptophan tryptophan domain-containing proteins. This protein shows 67% identity to the protein encoded by the chromosome 18 open reading frame 1 gene. Translation of STAG1/PMEPA1 mRNA in vitro showed two products of 36 and 39 kDa, respectively, suggesting that translation may initiate at more than one site. Comparison to genomic clones showed that STAG1/PMEPA1 was located on chromosome 20q13 between microsatellite markers D20S183 and D20S173 and spanned four exons and three introns. The upregulation of this gene in several solid tumors indicated that it may play an important role in tumorigenesis.

Identification and characterization of KLK14, a novel kallikrein serine protease gene located on human chromosome 19q13.4 and expressed in prostate and skeletal muscle.

The kallikreins are a subfamily of serine proteases encoded in human, mouse, and rat by highly conserved tightly clustered multigene families. Here we report the identification and characterization of KLK14, a novel kallikrein gene located within the human kallikrein locus at 19q13.4. KLK14 is approximately 5.4 kb in length spanning seven exons and, by Northern blot analysis, transcribes two alternative transcripts present only in prostate (1.5 kb) and skeletal muscle (1.9 kb). The protein product, K14, predicted to be a 251-amino-acid secreted serine protease with trypsin-like substrate specificity, is translated in vitro with a molecular mass of approximately 31 kDa. In situ hybridization revealed that, in prostate, KLK14 is expressed by both benign and malignant glandular epithelial cells, thus exhibiting an expression pattern similar to that of two other prostatic kallikreins, KLK2 and KLK3, which encode K2 and prostate-specific antigen, respectively.

Organization and chromosomal localization of the murine Testisin gene encoding a serine protease temporally expressed during spermatogenesis.

The recently characterized human serine protease, Testisin, is expressed on premeiotic testicular germ cells and is a candidate type II tumor suppressor for testicular cancer. Here we report the cloning, characterization and expression of the gene encoding mouse Testisin, Prss21. The murine Testisin gene comprises six exons and five introns and spans approximately 5 kb of genomic DNA with an almost identical structure to the human Testisin gene, PRSS21. The gene was localized to murine chromosome 17 A3.3-B; a region syntenic with the location of PRSS21 on human chromosome 16p13.3. Northern blot analyses of RNA from a range of adult murine tissues demonstrated a 1.3 kb mRNA transcript present only in testis. The murine Testisin cDNA shares 65% identity with human Testisin cDNA and encodes a putative pre-pro-protein of 324 amino acids with 80% similarity to human Testisin. The predicted amino-acid sequence includes an N-terminal signal sequence of 27 amino acids, a 27 amino-acid pro-region, a 251 amino-acid catalytic domain typical of a serine protease with trypsin-like specificity, and a C-terminal hydrophobic extension which is predicted to function as a membrane anchor. Immunostaining for murine Testisin in mouse testis demonstrated specific staining in the cytoplasm and on the plasma membrane of round and elongating spermatids. Examination of murine Testisin mRNA expression in developing sperm confirmed that the onset of murine Testisin mRNA expression occurred at approximately day 18 after birth, corresponding to the appearance of spermatids in the testis, in contrast to the expression of human Testisin in spermatocytes. These data identify the murine ortholog to human Testisin and demonstrate that the murine Testisin gene is temporally regulated during murine spermatogenesis.

Localization of the mosaic transmembrane serine protease corin to heart myocytes.

Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125-135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure.

Localization, expression and genomic structure of the gene encoding the human serine protease testisin.

Testisin is a recently identified human serine protease expressed by premeiotic testicular germ cells and is a candidate tumor suppressor for testicular cancer. Here, we report the characterization of the gene encoding testisin, designated PRSS21, and its localization on the short arm of human chromosome 16 (16p13.3) between the microsatellite marker D16S246 and the radiation hybrid breakpoint CY23HA. We have further refined the localization to cosmid 406D6 in this interval and have established that the gene is approximately 4. 5 kb in length, and contains six exons and five intervening introns. The structure of PRSS21 is very similar to the human prostasin gene (PRSS8) which maps nearby on 16p11.2, suggesting that these genes may have evolved through gene duplication. Sequence analysis showed that the two known isoforms of testisin are generated by alternative pre-mRNA splicing. A major transcription initiation site was identified 97 nucleotides upstream of the testisin translation start and conforms to a consensus initiator element. The region surrounding the transcription initiation site lacks a TATA consensus sequence, but contains a CCAAT sequence and includes a CpG island. The 5'-flanking region contains several consensus response elements including Sp1, AP1 and several testis-specific elements. Analysis of testisin gene expression in tumor cell lines shows that testisin is not expressed in testicular tumor cells but is aberrantly expressed in some tumor cell lines of non-testis origin. These data provide the basis for identifying potential genetic alterations of PRSS21 that may underlie both testicular abnormalities and tumorigenesis.

Tissue-specific expression patterns and fine mapping of the human kallikrein (KLK) locus on proximal 19q13.4.

The tissue or glandular kallikreins (KLK) are members of a highly conserved multigene family encoding serine proteases that are central to many biological processes. The rodent KLK families are large, highly conserved and clustered at one locus. The human KLK gene family is clustered on chromosome 19q13.3-13.4, and until recently consisted of just three members. However, recent studies have identified up to 11 new members of the KLK family that are less conserved than their rodent counterparts. Using a Southern blot and sequence analysis of 10 BACs and cosmids spanning approximately 400 kilobases (kb) either side of the original KLK 60-kb locus, we demonstrated that these genes also lie adjacent to this. We have also clarified the position of several microsatellite markers in relation to the extended KLK locus. Moreover, from Southern blot analysis of the cosmids and BACs with a degenerate oligonucleotide probe to the histidine-encoding region of serine proteases, we have shown that there are no other serine protease genes approximately 400 kb centromeric and 220 kb telomeric of the extended locus. We performed an extensive analysis of the expression patterns of these genes by poly(A)(+) RNA dot blot and reverse transcriptase-polymerase chain reaction analysis, and demonstrated a diverse pattern of expression. Of interest are clusters of genes with high prostate (KLK2-4) and pancreatic (KLK6-13) expression suggesting evolutionary conservation of elements conferring tissue specificity. From these findings, it is likely that the human KLK gene family consists of just 14 clustered genes within 300 kb and thus is of a comparable size to the rodent families (13-24 genes within 310 and 480 kb, respectively). In contrast to the rodent families, the newest members of the human KLK family are much less conserved in sequence (23-44% at the protein level) and appear to consist of at least four subfamilies. In addition, like the rat, these genes are expressed at varying levels in a diverse range of tissues although they exhibit quite distinct patterns of expression.

Cloning of the cDNA and localization of the gene encoding human NRBP, a ubiquitously expressed, multidomain putative adapter protein.

Adapter proteins modulate multiple signaling pathways by regulating the aggregation of other factors into signaling complexes. Here we have identified a novel human cDNA encoding NRBP, a multidomain putative adapter protein containing (i) two putative nuclear receptor binding motifs (LXXLL), (ii) a putative binding domain for Src homology-2 (SH2) domain containing proteins, (iii) a kinase-like domain, (iv) a bipartite nuclear localization signal, and (v) three sequences rich in glutamic acid, serine, proline, and threonine (PEST) residues. The NRBP mRNA transcript, of approximately 2.4 kb, was ubiquitously expressed in a wide range of normal human tissues and 15 human tumor cell lines. The NRBP cDNA is predicted to encode a polypeptide of 535 amino acids with a molecular mass of 59.8 kDa. Translation of NRBP mRNA in vitro reveals three translation products of 60, 51, and 43 kDa, suggesting that translation of NRBP may initiate at multiple sites. The NRBP gene was localized to human chromosome 2p23, near the location of the NCOA1 gene encoding the nuclear receptor coactivator, steroid receptor coactivator-1 (SRC-1). The features of NRBP predict a function as an adapter protein potentially linking signaling pathways involving nuclear receptors and SH2 domain containing proteins.

Testisin, a new human serine proteinase expressed by premeiotic testicular germ cells and lost in testicular germ cell tumors.

We have cloned and characterized a cDNA encoding a new human serine proteinase, testisin, that is abundantly expressed only in the testis and is lost in testicular tumors. The testisin cDNA was identified by homology cloning using degenerate primers directed at conserved sequence motifs within the catalytic regions of serine proteinases. It is 1073 nucleotides long, including 942 nucleotides of open reading frame and a 113-nucleotide 3' untranslated sequence. Northern and dot blot analyses of RNA from a range of normal human tissues revealed a 1.4-kb mRNA species that was present only in testis, which was not detected in eight of eight testicular tumors. Testisin cDNA is predicted to encode a protein of 314 amino acids, which consists of a 19-amino acid (aa) signal peptide, a 22-aa proregion, and a 273-aa catalytic domain, including a unique 17-aa COOH-terminal hydrophobic extension that is predicted to function as a membrane anchor. The deduced amino acid sequence of testisin shows 44% identity to prostasin and contains features that are typical of serine proteinases with trypsin-like substrate specificity. Antipeptide antibodies directed against the testisin polypeptide detected an immunoreactive testisin protein of Mr 35,000-39,000 in cell lysates from COS-7 cells that were transiently transfected with testisin cDNA. Immunostaining of normal testicular tissue showed that testisin was expressed in the cytoplasm and on the plasma membrane of premeiotic germ cells. No staining was detected in eight of eight germ cell-derived testicular tumors. In addition, the testisin gene was localized by fluorescence in situ hybridization to the short arm of human chromosome 16 (16p13.3), a region that has been associated with allellic imbalance and loss of heterozygosity in sporadic testicular tumors. These findings demonstrate a new cell surface serine proteinase, loss of which may have a direct or indirect role in the progression of testicular tumors of germ cell origin.