A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung.

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. WT and MR-deficient mice were exposed to lipopolysaccharide, after which the protein content in their lung epithelial lining fluid was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the epithelial lining fluid using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the TSP family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as urokinase plasminogen activator receptor-associated protein. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the TSP family.

The collagen receptor uPARAP/Endo180 regulates collectins through unique structural elements in its FNII domain.

C-type lectins that contain collagen-like domains are known as collectins. These proteins are present both in the circulation and in extravascular compartments and are central players of the innate immune system, contributing to first-line defenses against viral, bacterial, and fungal pathogens. The collectins mannose-binding lectin (MBL) and surfactant protein D (SP-D) are regulated by tissue fibroblasts at extravascular sites via an endocytic mechanism governed by urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180), which is also a collagen receptor. Here, we investigated the molecular mechanisms that drive the uPARAP-mediated cellular uptake of MBL and SP-D. We found that the uptake depends on residues within a protruding loop in the fibronectin type-II (FNII) domain of uPARAP that are also critical for collagen uptake. Importantly, however, we also identified FNII domain residues having an exclusive role in collectin uptake. We noted that these residues are absent in the related collagen receptor, the mannose receptor (MR or CD206), which consistently does not interact with collectins. We also show that the second C-type lectin-like domain (CTLD2) is critical for the uptake of SP-D, but not MBL, indicating an additional level of complexity in the interactions between collectins and uPARAP. Finally, we demonstrate that the same molecular mechanisms enable uPARAP to engage MBL immobilized on the surface of pathogens, thereby expanding the potential biological implications of this interaction. Our study reveals molecular details of the receptor-mediated cellular regulation of collectins and offers critical clues for future investigations into collectin biology and pathology.

Cellular uptake of collagens and implications for immune cell regulation in disease.

As the dominant constituent of the extracellular matrix (ECM), collagens of different types are critical for the structural properties of tissues and make up scaffolds for cellular adhesion and migration. Importantly, collagens also directly modulate the phenotypic state of cells by transmitting signals that influence proliferation, differentiation, polarization, survival, and more, to cells of mesenchymal, epithelial, or endothelial origin. Recently, the potential of collagens to provide immune regulatory signals has also been demonstrated, and it is believed that pathological changes in the ECM shape immune cell phenotype. Collagens are themselves heavily regulated by a multitude of structural modulations or by catabolic pathways. One of these pathways involves a cellular uptake of collagens or soluble collagen-like defense collagens of the innate immune system mediated by endocytic collagen receptors. This cellular uptake is followed by the degradation of collagens in lysosomes. The potential of this pathway to regulate collagens in pathological conditions is evident from the increased extracellular accumulation of both collagens and collagen-like defense collagens following endocytic collagen receptor ablation. Here, we review how endocytic collagen receptors regulate collagen turnover during physiological conditions and in pathological conditions, such as fibrosis and cancer. Furthermore, we highlight the potential of collagens to regulate immune cells and discuss how endocytic collagen receptors can directly regulate immune cell activity in pathological conditions or do it indirectly by altering the extracellular milieu. Finally, we discuss the potential collagen receptors utilized by immune cells to directly detect ECM-related changes in the tissues which they encounter.

The Collagen Receptor uPARAP in Malignant Mesothelioma: A Potential Diagnostic Marker and Therapeutic Target.

Malignant mesothelioma (MM) is a highly aggressive cancer with limited therapeutic options. We have previously shown that the endocytic collagen receptor, uPARAP, is upregulated in certain cancers and can be therapeutically targeted. Public RNA expression data display uPARAP overexpression in MM. Thus, to evaluate its potential use in diagnostics and therapy, we quantified uPARAP expression by immunohistochemical H-score in formalin-fixed paraffin-embedded bioptic/surgical human tissue samples and tissue microarrays. We detected pronounced upregulation of uPARAP in the three main MM subtypes compared to non-malignant reactive mesothelial proliferations, with higher expression in sarcomatoid and biphasic than in epithelioid MM. The upregulation appeared to be independent of patients' asbestos exposure and unaffected after chemotherapy. Using immunoblotting, we demonstrated high expression of uPARAP in MM cell lines and no expression in a non-malignant mesothelial cell line. Moreover, we showed the specific internalization of an anti-uPARAP monoclonal antibody by the MM cell lines using flow cytometry-based assays and confocal microscopy. Finally, we demonstrated the sensitivity of these cells towards sub-nanomolar concentrations of an antibody-drug conjugate formed with the uPARAP-directed antibody and a potent cytotoxin that led to efficient, uPARAP-specific eradication of the MM cells. Further studies on patient cohorts and functional preclinical models will fully reveal whether uPARAP could be exploited in diagnostics and therapeutic targeting of MM.

CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1-Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma.

BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects children and young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1-PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1-Prkaca fusion and monitored the mice for liver tumor development. METHODS: We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1-Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8-week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1-Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. RESULTS: Livers from 12 of the 15 mice given the vectors to induce the Dnajb1-Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1-Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1-Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. CONCLUSIONS: Using CRISPR/Cas9 technology, we found generation of the Dnajb1-Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1-PRKACA might be developed as therapeutics for this form of liver cancer.

Targeting a novel bone degradation pathway in primary bone cancer by inactivation of the collagen receptor uPARAP/Endo180.

In osteosarcoma, a primary mesenchymal bone cancer occurring predominantly in younger patients, invasive tumour growth leads to extensive bone destruction. This process is insufficiently understood, cannot be efficiently counteracted and calls for novel means of treatment. The endocytic collagen receptor, uPARAP/Endo180, is expressed on various mesenchymal cell types and is involved in bone matrix turnover during normal bone growth. Human osteosarcoma specimens showed strong expression of this receptor on tumour cells, along with the collagenolytic metalloprotease, MT1-MMP. In advanced tumours with ongoing bone degeneration, sarcoma cells positive for these proteins formed a contiguous layer aligned with the degradation zones. Remarkably, osteoclasts were scarce or absent from these regions and quantitative analysis revealed that this scarcity marked a strong contrast between osteosarcoma and bone metastases of carcinoma origin. This opened the possibility that sarcoma cells might directly mediate bone degeneration. To examine this question, we utilized a syngeneic, osteolytic bone tumour model with transplanted NCTC-2472 sarcoma cells in mice. When analysed in vitro, these cells were capable of degrading the protein component of surface-labelled bone slices in a process dependent on MMP activity and uPARAP/Endo180. Systemic treatment of the sarcoma-inoculated mice with a mouse monoclonal antibody that blocks murine uPARAP/Endo180 led to a strong reduction of bone destruction. Our findings identify sarcoma cell-resident uPARAP/Endo180 as a central player in the bone degeneration of advanced tumours, possibly following an osteoclast-mediated attack on bone in the early tumour stage. This points to uPARAP/Endo180 as a promising therapeutic target in osteosarcoma, with particular prospects for improved neoadjuvant therapy.

Crystal structures of the ligand-binding region of uPARAP: effect of calcium ion binding.

The proteins of the mannose receptor (MR) family share a common domain organization and have a broad range of biological functions. Urokinase plasminogen activator receptor-associated protein (uPARAP) (or Endo180) is a member of this family and plays an important role in extracellular matrix remodelling through interaction with its ligands, including collagens and urokinase plasminogen activator receptor (uPAR). We report the crystal structures of the first four domains of uPARAP (also named the ligand-binding region, LBR) at pH 7.4 in Ca(2+)-bound and Ca(2+)-free forms. The first domain (cysteine-rich or CysR domain) folds into a new and unique conformation different from the ß-trefoil fold of typical CysR domains. The so-called long loop regions (LLRs) of the C-type lectin-like domain (CTLD) 1 and 2 (the third and fourth domain) mediate the direct contacts between these domains. These LLRs undergo a Ca(2+)-dependent conformational change, and this is likely to be the key structural determinant affecting the overall conformation of uPARAP. Our results provide a molecular mechanism to support the structural flexibility of uPARAP, and shed light on the structural flexibility of other members of the MR family.

Early reversal cells in adult human bone remodeling: osteoblastic nature, catabolic functions and interactions with osteoclasts.

The mechanism coupling bone resorption and formation is a burning question that remains incompletely answered through the current investigations on osteoclasts and osteoblasts. An attractive hypothesis is that the reversal cells are likely mediators of this coupling. Their nature is a big matter of debate. The present study performed on human cancellous bone is the first one combining in situ hybridization and immunohistochemistry to demonstrate their osteoblastic nature. It shows that the Runx2 and CD56 immunoreactive reversal cells appear to take up TRAcP released by neighboring osteoclasts. Earlier preclinical studies indicate that reversal cells degrade the organic matrix left behind by the osteoclasts and that this degradation is crucial for the initiation of the subsequent bone formation. To our knowledge, this study is the first addressing these catabolic activities in adult human bone through electron microscopy and analysis of molecular markers. Periosteoclastic reversal cells show direct contacts with the osteoclasts and with the demineralized resorption debris. These early reversal cells show (1) ¾-collagen fragments typically generated by extracellular collagenases of the MMP family, (2) MMP-13 (collagenase-3) and (3) the endocytic collagen receptor uPARAP/Endo180. The prevalence of these markers was lower in the later reversal cells, which are located near the osteoid surfaces and morphologically resemble mature bone-forming osteoblasts. In conclusion, this study demonstrates that reversal cells colonizing bone surfaces right after resorption are osteoblast-lineage cells, and extends to adult human bone remodeling their role in rendering eroded surfaces osteogenic.

Loss of miR-10a activates lpo and collaborates with activated Wnt signaling in inducing intestinal neoplasia in female mice.

miRNAs are small regulatory RNAs that, due to their considerable potential to target a wide range of mRNAs, are implicated in essentially all biological process, including cancer. miR-10a is particularly interesting considering its conserved location in the Hox cluster of developmental regulators. A role for this microRNA has been described in developmental regulation as well as for various cancers. However, previous miR-10a studies are exclusively based on transient knockdowns of this miRNA and to extensively study miR-10a loss we have generated a miR-10a knock out mouse. Here we show that, in the Apc(min) mouse model of intestinal neoplasia, female miR-10a deficient mice develop significantly more adenomas than miR-10(+/+) and male controls. We further found that Lpo is extensively upregulated in the intestinal epithelium of mice deprived of miR-10a. Using in vitro assays, we demonstrate that the primary miR-10a target KLF4 can upregulate transcription of Lpo, whereas siRNA knockdown of KLF4 reduces LPO levels in HCT-116 cells. Furthermore, Klf4 is upregulated in the intestines of miR-10a knockout mice. Lpo has previously been shown to have the capacity to oxidize estrogens into potent depurinating mutagens, creating an instable genomic environment that can cause initiation of cancer. Therefore, we postulate that Lpo upregulation in the intestinal epithelium of miR-10a deficient mice together with the predominant abundance of estrogens in female animals mainly accounts for the sex-related cancer phenotype we observed. This suggests that miR-10a could be used as a potent diagnostic marker for discovering groups of women that are at high risk of developing colorectal carcinoma, which today is one of the leading causes of cancer-related deaths.

Complex determinants in specific members of the mannose receptor family govern collagen endocytosis.

Members of the well-conserved mannose receptor (MR) protein family have been functionally implicated in diverse biological and pathological processes. Importantly, a proposed common function is the internalization of collagen for intracellular degradation occurring during bone development, cancer invasion, and fibrosis protection. This functional relationship is suggested by a common endocytic capability and a candidate collagen-binding domain. Here we conducted a comparative investigation of each member's ability to facilitate intracellular collagen degradation. As expected, the family members uPARAP/Endo180 and MR bound collagens in a purified system and internalized collagens for degradation in cellular settings. In contrast, the remaining family members, PLA2R and DEC-205, showed no collagen binding activity and were unable to mediate collagen internalization. To pinpoint the structural elements discriminating collagen from non-collagen receptors, we constructed a series of receptor chimeras and loss- and gain-of-function mutants. Using this approach we identified a critical collagen binding loop in the suggested collagen binding region (an FN-II domain) in uPARAP/Endo180 and MR, which was different in PLA2R or DEC-205. However, we also found that an active FN-II domain was not a sufficient determinant to allow collagen internalization through these receptors. Nevertheless, this ability could be acquired by the transfer of a larger segment of uPARAP/Endo180 (the Cys-rich domain, the FN-II domain and two CTLDs) to DEC-205. These data underscore the importance of the FN-II domain in uPARAP/Endo180 and MR-mediated collagen internalization but at the same time uncover a critical interplay with flanking domains.

Monitoring mammary tumor progression and effect of tamoxifen treatment in MMTV-PymT using MRI and magnetic resonance spectroscopy with hyperpolarized [1-13C]pyruvate.

PURPOSE: To use dynamic magnetic resonance spectroscopy (MRS) of hyperpolarized (13)C-pyruvate to follow the progress over time in vivo of breast cancer metabolism in the MMTV-PymT model, and to follow the response to the anti-estrogen drug tamoxifen. METHODS: Tumor growth was monitored by anatomical MRI by measuring tumor volumes. Dynamic MRS of hyperpolarized (13)C was used to measure an "apparent" pyruvate-to-lactate rate constant (kp) of lactate dehydrogenase (LDH) in vivo. Further, ex vivo pathology and in vitro LDH initial reaction velocity were evaluated. RESULTS: Tamoxifen significantly halted the tumor growth measured as tumor volume by MRI. In the untreated animals, kp correlated with tumor growth. The kP was somewhat but not significantly lower in the treated group. Studies in vitro confirmed the effects of tamoxifen on tumor growth, and here the LDH reaction velocity was reduced significantly in the treated group. CONCLUSION: These hyperpolarized (13)C MRS findings indicate that tumor metabolic changes affects kP. The measured kp did not relate to treatment response to the same extent as did tumor growth, histological evaluation, and in vitro determination of LDH activity.

Targeting a single function of the multifunctional matrix metalloprotease MT1-MMP: impact on lymphangiogenesis.

The group of matrix metalloproteases (MMPs) is responsible for multiple processes of extracellular matrix remodeling in the healthy body but also for matrix and tissue destruction during cancer invasion and metastasis. The understanding of the contributions from each individual MMP, both in healthy and pathological events, has been complicated by the lack of specific inhibitors and the fact that some of the potent MMPs are multifunctional enzymes. These factors have also hampered the setup of therapeutic strategies targeting MMP activity. A tempting target is the membrane-associated MT1-MMP, which has well-documented importance in matrix degradation but which takes part in more than one pathway in this regard. In this report, we describe the selective targeting of a single function of this enzyme by means of a specific monoclonal antibody against MT1-MMP, raised in an MT1-MMP knock-out mouse. The antibody blocks the enzyme ability to activate proMMP-2 without interfering with the collagenolytic function or the general proteolytic activity of MT1-MMP. Using this antibody, we have shown that the MT1-MMP-catalyzed activation of proMMP-2 is involved in the outgrowth of cultured lymphatic endothelial cells in a collagen matrix in vitro, as well as in lymphatic vessel sprouting assayed ex vivo. This is the first example of the complete inactivation of a single function of a multifunctional MMP and the use of this strategy to pursue its role.

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently.

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.

Antibody-Drug Conjugates: The Dynamic Evolution from Conventional to Next-Generation Constructs.

Introduced almost two decades ago, ADCs have marked a breakthrough in the targeted therapy era, providing clinical benefits to many cancer patients. While the inherent complexity of this class of drugs has challenged their development and broad application, the experience gained from years of trials and errors and recent advances in construct design and delivery have led to an increased number of ADCs approved or in late clinical development in only five years. Target and payload diversification, along with novel conjugation and linker technologies, are at the forefront of next-generation ADC development, renewing hopes to broaden the scope of these targeted drugs to difficult-to-treat cancers and beyond. This review highlights recent trends in the ADC field, focusing on construct design and mechanism of action and their implications on ADCs' therapeutic profile. The evolution from conventional to innovative ADC formats will be illustrated, along with some of the current hurdles, including toxicity and drug resistance. Future directions to improve the design of next-generation ADCs will also be presented.

Endocytic collagen degradation: a novel mechanism involved in protection against liver fibrosis.

Fibrosis of the liver and its end-stage, cirrhosis, represent major health problems worldwide. In these fibrotic conditions, activated fibroblasts and hepatic stellate cells display a net deposition of collagen. This collagen deposition is a major factor leading to liver dysfunction, thus making it crucially important to understand both the collagen synthesis and turnover mechanisms in this condition. Here we show that the endocytic collagen receptor, uPARAP/Endo180, is a major determinant in governing the balance between collagen deposition and degradation. Cirrhotic human livers displayed a marked up-regulation of uPARAP/Endo180 in activated fibroblasts and hepatic stellate cells located close to the collagen deposits. In a hepatic stellate cell line, uPARAP/Endo180 was shown to be active in, and required for, the uptake and intracellular degradation of collagen. To evaluate the functional importance of this collagen receptor in vivo, liver fibrosis was induced in uPARAP/Endo180-deficient mice and littermate wild-type mice by chronic CCl(4) administration. A strong up-regulation of uPARAP/Endo180 was observed in wild-type mice, and a quantitative comparison of collagen deposits in the two groups of mice clearly revealed a fibrosis protective role of uPARAP/Endo180. This effect appeared to directly reflect the activity of the collagen receptor, since no compensatory events were noted when comparing the mRNA expression profiles of the two groups of mice in an array system focused on matrix-degrading components. This function of uPARAP/Endo180 defines a novel role of intracellular collagen turnover in fibrosis protection.

Targeted delivery of alcohol-containing payloads with antibody-drug conjugates.

We herein describe the cell-specific release of alcohol-containing payloads via a sulfatase-sensitive linker in antibody-drug conjugates (ADCs). The linker shows efficient sulfatase-mediated release and high stability in human and mouse plasma. In vitro evaluation demonstrates potent antigen dependent toxicity towards breast cancer cell lines.

A novel functional role of collagen glycosylation: interaction with the endocytic collagen receptor uparap/ENDO180.

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation.

The non-phagocytic route of collagen uptake: a distinct degradation pathway.

The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include ß1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. ß1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

Uncovering mediators of collagen degradation in the tumor microenvironment.

Increased remodeling of the extracellular matrix in malignant tumors has been shown to correlate with tumor aggressiveness and a poor prognosis. This remodeling involves degradation of the original extracellular matrix (ECM) and deposition of a new tumor-supporting ECM. The main constituent of the ECM is collagen and collagen turnover mainly occurs in a sequential manner, where initial proteolytic cleavage of the insoluble fibers is followed by cellular internalization of large well-defined collagen fragments for lysosomal degradation. However, despite extensive research in the field, a lack of consensus on which cell types within the tumor microenvironment express the involved proteases still exists. Furthermore, the relative contribution of different cell types to collagen internalization is not well-established. Here, we developed quantitative ex vivo collagen degradation assays and show that the proteases responsible for the initial collagen cleavage in two murine syngeneic tumor models are matrix metalloproteinases produced by cancer-associated fibroblasts and that collagen degradation fragments are endocytosed primarily by tumor-associated macrophages and cancer-associated fibroblasts from the tumor stroma. Using tumors from mannose receptor-deficient mice, we show that this receptor is essential for collagen-internalization by tumor-associated macrophages. Together, these findings identify the cell types responsible for the entire collagen degradation pathway, from initial cleavage to endocytosis of fragments for intracellular degradation.

The endocytic receptor uPARAP is a regulator of extracellular thrombospondin-1.

Thrombospondin-1 (TSP-1) is a matricellular protein with a multitude of functions in the pericellular and extracellular environment. We report a novel pathway for the regulation of extracellular TSP-1, governed by the endocytic collagen receptor, uPARAP (urokinase plasminogen activator receptor-associated protein; MRC2 gene product, also designated Endo180, CD280). First, using a novel proteomic approach for unbiased identification of ligands for endocytosis, we identify TSP-1 as a candidate ligand for specific uptake by uPARAP. We then show that uPARAP can efficiently internalize TSP-1 for lysosomal degradation, that this capability is not shared by other, closely related endocytic receptors and that uPARAP serves to regulate the extracellular levels of TSP-1 in vitro. Using wild type and uPARAP null mice, we also demonstrate uPARAP-mediated endocytosis of TSP-1 in dermal fibroblasts in vivo. Unlike other uPARAP ligands, the interaction with TSP-1 is sensitive to heparin and the responsible molecular motifs in uPARAP are overlapping, but not identical with those governing the interaction with collagens. Finally, we show that uPARAP can also mediate the endocytosis of TSP-2, a thrombospondin closely related to TSP-1, but not the more distantly related members of the same protein family, TSP-3, -4 and -5. These findings indicate that the role of uPARAP in ECM remodeling is not limited to the uptake of collagen for degradation but also includes an orchestrator function in the regulation of thrombospondins with numerous downstream effects. This is likely to be an important factor in the physiological and pathological roles of uPARAP in bone biology, fibrosis and cancer. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD031272.