Novel human lymph node-derived matrix supports the adhesion of metastatic oral carcinoma cells.

BACKGROUND: 3D culture is increasingly used in cancer research, as it allows the growth of cells in an environment that mimics in vivo conditions. Metastases are the primary cause of morbidity and mortality in cancer patients, and solid tumour metastases are mostly located in lymph nodes. Currently, there are no techniques that model the pre-metastatic lymph node microenvironment in vitro. In this study, we prepared a novel extracellular matrix, Lymphogel, which is derived from lymph nodes, mimicking the tumour microenvironment (TME) of metastatic carcinoma cells. We tested the suitability of the new matrix in various functional experiments and compared the results with those obtained using existing matrices. METHODS: We used both commercial and patient-derived primary and metastatic oral tongue squamous cell carcinoma (OTSCC) cell lines. We characterized the functional differences of these cells using three different matrices (human uterine leiomyoma-derived Myogel, human pre-metastatic neck lymph node-derived Lymphogel (h-LG), porcine normal neck lymph node-derived Lymphogel (p-LG) in proliferation, adhesion, migration and invasion assays. We also performed proteomic analyses to compare the different matrices in relation to their functional properties. RESULTS: OTSCC cells exhibited different adhesion and invasion patterns depending on the matrix. Metastatic cell lines showed improved ability to adhere to h-LG, but the effects of the matrices on cell invasion fluctuated non-significantly between the cell lines. Proteomic analyses showed that the protein composition between matrices was highly variable; Myogel contained 618, p-LG 1823 and h-LG 1520 different proteins. The comparison of all three matrices revealed only 120 common proteins. Analysis of cellular pathways and processes associated with proteomes of each matrix revealed similarities of Myogel with h-LG but less with p-LG. Similarly, p-LG contained the least adhesion-related proteins compared with Myogel and h-LG. The highest number of unique adhesion-related proteins was present in h-LG. CONCLUSIONS: We demonstrated that human pre-metastatic neck lymph node-derived matrix is suitable for studying metastatic OTSCC cells. As a whole-protein extract, h-LG provides new opportunities for in vitro carcinoma cell culture experiments.

Loss of Caveolin-1 Expression in Tumor Cells is Associated with Increased Aggressiveness and Cell Invasion in Oral Squamous Cell Carcinoma.

BACKGROUND: Changes in Caveolin-1 (CAV-1) expression are related to tumorigenesis. The aim of this study was to evaluate the role of CAV-1 in tumor progression in oral squamous cell carcinoma (SCC) tissue samples and the effect of CAV-1 silencing on two oral tongue SCC (OTSCC) cell lines (SCC-25, from a primary tumor, and HSC-3 from lymph node metastases). METHODS: Mycroarray hybridization, mRNA expression, and immunohistochemistry were performed on OSCC tissue samples and corresponding non-tumoral margin tissues. The effects of CAV-1 silencing (siCAV-1) on cell viability, membrane fluidity, on the expression of epithelial to mesenchymal transition (EMT) markers and on cell migration and invasion capacity of OTSCC cell lines were evaluated. RESULTS: Microarray showed a greater CAV-1 expression (1.77-fold) in OSCC tumors than in non-tumoral tissues and 2.0-fold more in less aggressive OSCCs. However, significant differences in CAV-1 gene expression were not seen between tumors and non-tumoral margins nor CAV-1 with any clinicopathological parameters. CAV-1 protein was localized both in carcinoma and in spindle cells of the tumor microenvironment (TME), and CAV-1 positive TME cells were associated with smaller/more aggressive tumors, independent of the carcinoma cells' expression. Silencing of CAV-1 increased cell viability only in SCC-25 cells. It also stimulated the invasion of HSC-3 cells and increased ECAD and BCAT mRNA in these cells; however, the protein levels of the EMT markers were not affected. CONCLUSION: Decreased expression of CAV-1 by tumor cells in OSCC and an increase in the TME were associated with increased cell invasiveness and tumor aggressiveness.

Detection of herpes simplex virus in oral tongue squamous cell carcinoma.

Introduction: Oral tongue squamous cell carcinoma (OTSCC) is the most common cancer of the oral cavity. Contradictory results have been observed on the involvement of herpes simplex virus 1 (HSV-1) in oral squamous cell carcinomas. Here, we aimed to study the predominance of HSV-1 or HSV-2 in oral HSV infections and to investigate the presence of HSV-1 in OTSCC and its effect on carcinoma cell viability and invasion. Methods: The distribution of HSV types one and two in diagnostic samples taken from suspected oral HSV infections was determined from the Helsinki University Hospital Laboratory database. We then analysed 67 OTSCC samples for HSV-1 infection using immunohistochemical staining. We further tested the effects of HSV-1 using six concentrations (0.00001-1.0 multiplicity of infection [MOI]) on viability and two concentrations (0.001 and 0.1 MOI) on invasion of highly invasive metastatic HSC-3 and less invasive primary SCC-25 OTSCC cell lines using MTT and Myogel-coated Transwell invasion assays. Results: Altogether 321 oropharyngeal samples were diagnosed positive for HSV during the study period. HSV-1 was the predominant (97.8%) HSV type compared with HSV-2 (detected in 2.2% of samples). HSV-1 was also detected in 24% of the OTSCC samples and had no association with patient survival or recurrence. OTSCC cells were viable even after 6 days with low viral load (0.00001, 0.0001, 0.001 MOI) of HSV-1. In both cell lines, 0.001 MOI did not affect cell invasion. However, 0.1 MOI significantly reduced cell invasion in HSC-3 cells. Discussion: HSV-1 infection is predominant compared with HSV-2 in the oral cavity. HSV-1 is detected in OTSCC samples without clinical significance, and OTSCC cell survival or invasion was not affected at low doses of HSV-1.

Overexpression of heat-shock protein 47 impacts survival of patients with oral squamous cell carcinoma.

BACKGROUND: The expression of heat-shock protein 47 (HSP47) has been linked to collagen synthesis control and implicated in fibrotic disorders, but more recent studies have demonstrated its role in solid tumors. In this study, we explored the prognostic impact of HSP47 in oral squamous cell carcinomas (OSCC) and determined the in vitro effects of its loss-of-function on viability, proliferation, migration, invasion, and resistance to cisplatin of OSCC cells. METHODS: The HSP47 expression in tumor samples was assessed by immunohistochemistry in two independent cohorts totaling 339 patients with OSCC, and protein levels were associated with clinicopathological features and survival outcomes. The OSCC cell lines HSC3 and SCC9 were transduced with lentivirus expressing short hairpin RNA to stably silence HSP47 and used in assays to measure cellular viability, proliferation, migration, and invasion. RESULTS: HSP47 was overexpressed in OSCC samples, and its overexpression was significantly and independently associated with poor disease-specific survival and shortened disease-free survival in both OSCC cohorts. The knockdown of HSP47 showed no effects on cell viability or cisplatin sensitivity, but impaired significantly proliferation, migration, and invasion of OSCC cells, with stronger effects on SCC9 cells. CONCLUSION: Our results show a significant prognostic impact of HSP47 overexpression in OSCC and reveal that HSP47 inhibition impairs the proliferation, migration, and invasion of OSCC cells. HSP47 may represent a potential therapeutic target for OSCC.

Cholesterol depletion affects caveolin-1 expression, migration and invasion of oral tongue squamous cell carcinoma cell lines.

INTRODUCTION: Cholesterol is a key lipid molecule within cell membranes. This is especially true in cavelolas, invaginated membrane nanodomains, which present the protein caveolin-1 (CAV-1). It is important to note that this structure is involved in many cell signalling pathways. Additionally, high cholesterol is seen in different tumor types but little is known in regards to oral tongue squamous cell carcinoma (OTSCC). The aim of this study was to evaluate the influence of cholesterol depletion on primary (SCC-25) and metastatic (HSC-3) OTSCC cell lines. MATERIALS AND METHODS: Cell membrane fluidity, cell viability, gene and protein expression of CAV-1 and of epithelial-mesenchymal transition (EMT) markers, cell migration in Myogel and invasion-myoma assay were evaluated after cholesterol depletion with methyl-ß-cyclodextrin (MßCD - 7.5, 10 or 15 mM) RESULTS: Decreased cell viability and increased membrane fluidity of SCC-25 cells was seen with cholesterol depletion but cell viability was less affected and there was no effect on membrane fluidity in HSC-3. Cholesterol depletion also decreased CAV-1 at 6 h but increased it after 24 h.; both epithelial and mesenchymal EMT genes were upregulated after 6 h, followed by downregulation at 24 h in SCC-25. In HSC-3, CAV-1 was downregulated, and E-cadherin gene (ECAD) was upregulated at 6 h. Only the protein ß-catenin in SCC-25 was affected, and cell migration of both cell lines was decreased, affecting SCC-25 more intensely. The invasive capacity within human myoma organotypic model was increased in SCC-25 and decreased in HSC-3. CONCLUSION: Cholesterol depletion affects CAV-1 and ECAD inversely. This affect also depends on cell type since the invasive capacity was augmented in primary cells while decreased in metastatic cells.

Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment.

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients. Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

Prognostic value of dysadherin in cancer: A systematic review and meta-analysis.

Cancer is a leading cause of death worldwide and novel prognostic factors are reported with increasing numbers. Systematic reviews and meta-analyses on cumulative research data are crucial in estimating the true prognostic value of proposed factors. Dysadherin (FXYD Domain Containing Ion Transport Regulator 5; FXYD5) is a cell membrane glycoprotein that modulates Na+, K+-ATPase activity and cell-cell adhesion. It is abundantly expressed in a variety of cancer cells, but only in a limited number of normal cells and its levels are increased in many different tumor types. The expression or level of dysadherin has been suggested as an independent predictor for metastasis and poor prognosis by number of studies, yet we lack a definitive answer. In this study, we systematically evaluated the prognostic value of dysadherin in cancer and summarized the current knowledge on the subject. PubMed, Scopus, Web of Science and relevant clinical trial and preprint databases were searched for relevant publications and PRISMA and REMARK guidelines were applied in the process. After a careful review, a total of 23 original research articles were included. In each study, dysadherin was pointed as a marker for poor prognosis. Meta-analyses revealed 3- and 1.5-fold increases in the risk of death (fixed effects HR 3.08, 95% CI 1.88-5.06, RR 1.47, 95% CI 1.06-2.05 on overall survival, respectively) for patients with high (>50%) tumoral FXYD5 level. In many studies, a connection between dysadherin expression or level and metastatic behavior of the cancer as well as inverse correlation with E-cadherin level were reported. Thus, we conclude that dysadherin might be a useful prognostic biomarker in the assessment of disease survival of patients with solid tumors.

Effects of Fermented Wheat Germ Extract on Oral Cancer Cells: An In Vitro Study.

Oral carcinoma is one of the most aggressive cancers, and despite the advances in the therapy, its mortality is still high. An attention in cancer treatment has focused on natural compounds due to their potential beneficial effects on human health. In this study, the effects of dietary supplement Fermented Wheat Germ Extract (FWGE) on oral tongue squamous cell carcinoma (OTSCC) cells were investigated In Vitro using three cell lines (HSC-3, SAS, SCC-25) with variable aggressiveness. The cell viability was significantly decreased by the treatment with high concentration of FWGE in every cell line. Regarding migration and invasion, HSC-3 and SCC-25 cells were most sensitive to FWGE since their movement was significantly reduced with 5 and 10 mg/ml FWGE, while SAS was inhibited only with 10 mg/ml FWGE. Chemotherapeutic compounds (cisplatin and 5-fluorouracil) significantly reduced all OTSCC cells viability. Importantly, combination of these drugs with 10 mg/ml FWGE significantly decreased the cell viability compared to the treatment with the chemotherapeutics or FWGE alone. Based on these In Vitro experiments, the use of FWGE seems to improve the anticancer effects on OTSCC cells. Further In Vivo and clinical studies should be conducted to verify the positive effects of FWGE for OTSCC patients.

Stress induced phosphoprotein 1 overexpression controls proliferation, migration and invasion and is associated with poor survival in oral squamous cell carcinoma.

Objective: Although there have been remarkable achievements in the molecular landscape of oral squamous cell carcinoma (OSCC) in recent years, bringing advances in the understanding of its pathogenesis, development and progression, little has been applied in the prognosis and choosing the optimal treatment. In this study, we explored the influence of the stress induced phosphoprotein 1 (STIP1), which is frequently reported to be highly expressed in many cancers, in OSCCs. Methods: STIP1 expression was assessed in the TCGA database and in two independent cohorts by immunohistochemistry. Knockdown strategy was applied in OSCC cell lines to determine the impact of STIP1 on viability, proliferation, migration and invasion. The zebrafish model was applied for studying tumor formation and metastasis in vivo. The association of STIP1 and miR-218-5p was explored by bioinformatics and mimics transfection. Results: STIP1 was highly expressed in OSCCs and significantly associated with shortened survival and higher risk of recurrence. STIP1 down-regulation decreased proliferation, migration and invasion of tumor cells, and reduced the number of metastases in the Zebrafish model. STIP1 and miR-218-5p were inversely expressed, and the transfection of miR-218-5p mimics into OSCC cells decreased STIP1 levels as well as proliferation, migration and invasion. Conclusion: Our findings show that STIP1 overexpression, which is inversely associated with miR-218-5p levels, contributes to OSCC aggressiveness by controlling proliferation, migration and invasion and is a determinant of poor prognosis.

High-throughput compound screening identifies navitoclax combined with irradiation as a candidate therapy for HPV-negative head and neck squamous cell carcinoma.

Conventional chemotherapeutic agents are nonselective, often resulting in severe side effects and the development of resistance. Therefore, new molecular-targeted therapies are urgently needed to be integrated into existing treatment regimens. Here, we performed a high-throughput compound screen to identify a synergistic interaction between ionizing radiation and 396 anticancer compounds. The assay was run using five human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) cell lines cultured on the human tumor-derived matrix Myogel. Our screen identified several compounds with strong synergistic and antagonistic effects, which we further investigated using multiple irradiation doses. Navitoclax, which emerged as the most promising radiosensitizer, exhibited synergy with irradiation regardless of the p53 mutation status in all 13 HNSCC cell lines. We performed a live cell apoptosis assay for two representative HNSCC cell lines to examine the effects of navitoclax and irradiation. As a single agent, navitoclax reduced proliferation and induced apoptosis in a dose-dependent manner, whereas the navitoclax-irradiation combination arrested cell cycle progression and resulted in substantially elevated apoptosis. Overall, we demonstrated that combining navitoclax with irradiation resulted in synergistic in vitro antitumor effects in HNSCC cell lines, possibly indicating the therapeutic potential for HNSCC patients.

SLC4A2 anion exchanger promotes tumour cell malignancy via enhancing net acid efflux across golgi membranes.

Proper functioning of each secretory and endocytic compartment relies on its unique pH micro-environment that is known to be dictated by the rates of V-ATPase-mediated H+ pumping and its leakage back to the cytoplasm via an elusive "H+ leak" pathway. Here, we show that this proton leak across Golgi membranes is mediated by the AE2a (SLC4A2a)-mediated bicarbonate-chloride exchange, as it is strictly dependent on bicarbonate import (in exchange for chloride export) and the expression level of the Golgi-localized AE2a anion exchanger. In the acidic Golgi lumen, imported bicarbonate anions and protons then facilitate a common buffering reaction that yields carbon dioxide and water before their egress back to the cytoplasm via diffusion or water channels. The flattened morphology of the Golgi cisternae helps this process, as their high surface-volume ratio is optimal for water and gas exchange. Interestingly, this net acid efflux pathway is often upregulated in cancers and established cancer cell lines, and responsible for their markedly elevated Golgi resting pH and attenuated glycosylation potential. Accordingly, AE2 knockdown in SW-48 colorectal cancer cells was able to restore these two phenomena, and at the same time, reverse their invasive and anchorage-independent growth phenotype. These findings suggest a possibility to return malignant cells to a benign state by restoring Golgi resting pH.

Fluctuating role of antimicrobial peptide hCAP18/LL­37 in oral tongue dysplasia and carcinoma.

Oral tongue squamous cell carcinoma (OTSCC), the most common cancer in the oral cavity, is aggressive and its incidence is increasing globally. Human host defense cationic antimicrobial peptide­18/antimicrobial peptide leucine­leucine­37 (hCAP18/LL­37) plays a complex role in various types of cancers. In the present study, we characterized the effects of exogenous LL­37 on three OTSCC cell lines and determined the expression of hCAP18/LL­37 in oral dysplastic and OTSCC patient samples. Our data revealed that LL­37, especially in high doses, mostly reduced the proliferation of OTSCC cells, but the effect was fluctuating. However, LL­37 stimulated the migration and invasion of OTSCC cells. The high dose of LL­37 also increased the amount of total epidermal growth factor receptor (EGFR) probably due to stabilization of the receptor to the plasma membrane. However, activation of EGFR downstream pathways was mostly decreased. Our immunohistochemical analysis showed that the hCAP18/LL­37 expression was higher in normal/mild dysplasia than in moderate/severe dysplasia and OTSCC. The hCAP18/LL­37 expression did not correlate with clinicopathological features or outcome of OTSCC patients. Our data suggest that LL­37 has a fluctuating effect on proliferation, migration and invasion of OTSCC cells, but it does not seem to play a role in the progression of OTSCC.

Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells.

Pro-tumorigenic activities of matrix metalloproteinase (MMP) 9 have been linked to many cancers, but recently the tumour-suppressing role of MMP9 has also been elucidated. The multifaceted evidence on this subject prompted us to examine the role of MMP9 in the behaviour of oral tongue squamous cell carcinoma (OTSCC) cells. We used gelatinase-specific inhibitor, CTT2, and short hairpin (sh) RNA gene silencing to study the effects of MMP9 on proliferation, motility and invasion of an aggressive OTSCC cell line, HSC-3. We found that the migration and invasion of HSC-3 cells were increased by CTT2 and shRNA silencing of MMP9. Proliferation, in turn, was decreased by MMP9 inhibition. Furthermore, arresten-overexpressing HSC-3 cells expressed increased levels of MMP9, but exhibited decreased motility compared with controls. Interestingly, these cells restored their migratory capabilities by CTT2 inhibition of MMP9. Hence, although higher MMP9 expression could give rise to an increased tumour growth in vivo due to increased proliferation, in some circumstances, it may participate in yet unidentified molecular mechanisms that reduce the cell movement in OTSCC.

Human Tumor-Derived Matrix Improves the Predictability of Head and Neck Cancer Drug Testing.

In vitro cancer drug testing carries a low predictive value. We developed the human leiomyoma-derived matrix "Myogel" to better mimic the human tumor microenvironment (TME). We hypothesized that Myogel could provide an appropriate microenvironment for cancer cells, thereby allowing more in vivo-relevant drug testing. We screened 19 anticancer compounds, targeting the epidermal growth factor receptor (EGFR), MEK, and PI3K/mTOR on 12 head and neck squamous cell carcinoma (HNSCC) cell lines cultured on plastic, mouse sarcoma-derived Matrigel (MSDM), and Myogel. We applied a high-throughput drug screening assay under five different culturing conditions: cells in two-dimensional (2D) plastic wells and on top or embedded in Matrigel or Myogel. We then compared the efficacy of the anticancer compounds to the response rates of 19 HNSCC monotherapy clinical trials. Cancer cells on top of Myogel responded less to EGFR and MEK inhibitors compared to cells cultured on plastic or Matrigel. However, we found a similar response to the PI3K/mTOR inhibitors under all culturing conditions. Cells grown on Myogel more closely resembled the response rates reported in EGFR-inhibitor monotherapy clinical trials. Our findings suggest that a human tumor matrix improves the predictability of in vitro anticancer drug testing compared to current 2D and MSDM methods.

Effects of ionizing radiation and HPSE1 inhibition on the invasion of oral tongue carcinoma cells on human extracellular matrices in vitro.

Chemoradiation is an established approach in the treatment of advanced oral tongue squamous cell carcinoma (OTSCC), but therapy may cause severe side-effects due to signal interchanges between carcinoma and the tumour microenvironment (TME). In this study, we examined the potential use of our human 3D myoma disc and Myogel models in in vitro chemoradiation studies by analysing the effects of ionizing radiation (IR) and the combined effect of heparanase I (HPSE1) inhibitors and IR on OTSCC cell proliferation, invasion and MMP-2 and - 9 production. Finally, we analysed the long-term effects of IR by studying clones of previously irradiated and invaded HSC-3 cells. We found that in both human uterine leiomyoma-based extracellular matrix models IR inhibited the invasion of HSC-3 cells, but blocking HPSE1 activity combined with IR induced their invasion. Low doses of IR increased MMP expression and initiated epithelial-mesenchymal transition in cells cultured on myoma discs. We conclude that myoma models offer consistent methods for testing human carcinoma cell invasion and phenotypic changes during chemoradiation treatment. In addition, we showed that IR had long-term effects on MMP-2 and - 9, which might elicit different HSC-3 invasion responses when cells were under the challenge of HPSE1 inhibitors and IR.

Inhibitory effects of serum from sepsis patients on epithelial cell migration in vitro: a case control study.

BACKGROUND: Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis sera as well as the effects of cytokines, growth factors and exosomes of sepsis sera treated normal fibroblasts (NF) on keratinocyte migration and proliferation in vitro. METHODS: Serum samples were taken on days 1, 4, and 9 from 44 patients diagnosed with severe sepsis, and from 14 matching healthy controls. We evaluated the effects of sepsis serum with or without TNF-α, EGF, EGF receptor inhibitor or exosomes of sepsis sera treated NF on human keratinocyte (HaCaT) proliferation (BrdU assay), viability (MTT assay), and migration (horizontal wound healing model). Cytokine levels of sepsis and healthy sera were measured by multiplex assay. Comparisons between groups were carried out using SPSS statistics and P < 0.05 was considered significant. RESULTS: Severe-sepsis sera collected on days 1, 4, and 9 reduced keratinocyte proliferation by 6% (P = 0.005), 20% (P = 0.001), and 18% (P = 0.002), respectively, compared to control sera. Cell viability in cultures exposed to sepsis sera from days 4 and 9 was reduced by 38% (P = 0.01) and 58% (P < 0.001), respectively. Open-surface wounds exposed to sepsis sera from days 1 and 4 were larger than those exposed to sera from healthy controls (60 vs. 31%, P = 0.034 and 66 vs. 31%, P = 0.023, respectively). Exosomes of sepsis or healthy sera treated NF inhibited keratinocyte migration. We detected higher serum levels of cytokines TNF-α (5.7 vs. 0.7 pg/ml, P < 0.001), IL-6 (24.8 vs. 3.8 pg/ml, P < 0.001), IL-10 (30.0 vs. 11.9 pg/ml, P = 0.040), and VEGF (177.9 vs. 48.1 pg/ml, P = 0.018) in sepsis sera. Levels of EGF were significantly lower in sepsis sera than in that of healthy controls (6.5 vs. 115.6 pg/ml, P < 0.001). Sepsis serum supplemented with EGF 5 ng/ml and TNF-α in all concentrations improved keratinocyte migration. CONCLUSIONS: Keratinocyte viability, proliferation and migration were reduced in severe sepsis in vitro. Exosomes from NF added in healthy or sepsis serum media inhibited keratinocyte migration. Decreased levels of EGF in sepsis sera may partially explain the delay of wound healing with severe-sepsis patients. Increased levels of TNF-α in sepsis sera do not explain diminished keratinocyte migration.

Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis.

Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.

Lysyl hydroxylase 3 modifies lysine residues to facilitate oligomerization of mannan-binding lectin.

Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers.

The activities of lysyl hydroxylase 3 (LH3) regulate the amount and oligomerization status of adiponectin.

Lysyl hydroxylase 3 (LH3) has lysyl hydroxylase, galactosyltransferase, and glucosyltransferase activities, which are sequentially required for the formation of glucosylgalactosyl hydroxylysines in collagens. Here we demonstrate for the first time that LH3 also modifies the lysine residues in the collagenous domain of adiponectin, which has important roles in glucose and lipid metabolism and inflammation. Hydroxylation and, especially, glycosylation of the lysine residues of adiponectin have been shown to be essential for the formation of the more active high molecular weight adiponectin oligomers and thus for its function. In cells that totally lack LH3 enzyme, the galactosylhydroxylysine residues of adiponectin were not glucosylated to glucosylgalactosylhydroxylysine residues and the formation of high and middle molecular weight adiponectin oligomers was impaired. Circulating adiponectin levels in mutant mice lacking the lysyl hydroxylase activity of LH3 were significantly reduced, which indicates that LH3 is required for complete modification of lysine residues in adiponectin and the loss of some of the glycosylated hydroxylysine residues severely affects the secretion of adiponectin. LH mutant mice with reduced adiponectin level showed a high fat diet-induced increase in glucose, triglyceride, and LDL-cholesterol levels, hallmarks of the metabolic syndrome in humans. Our results reveal the first indication that LH3 is an important regulator of adiponectin biosynthesis, secretion and activity and thus might be a potential candidate for therapeutic applications in diseases associated with obesity and insulin resistance.

Dimerization of human lysyl hydroxylase 3 (LH3) is mediated by the amino acids 541-547.

Lysyl hydroxylases (LH), which catalyze the post-translational modifications of lysines in collagen and collagen-like proteins, function as dimers. However, the amino acids responsible for dimerization and the role of dimer formation in the enzymatic activities of LH have not yet been identified. We have localized the region responsible for the dimerization of lysyl hydroxylase 3 (LH3), a multifunctional enzyme of collagen biosynthesis, to a sequence of amino acids between the glycosyltransferase activity and the lysyl hydroxylase activity domains. This area is covered by amino acids 541-547 in human LH3, but contains no cysteine residues. The region is highly conserved among LH isoforms, and is also involved in the dimerization of LH1 subunits. Dimerization is required for the LH activity of LH3, whereas it is not obligatory for the glycosyltransferase activities. In order to determine whether complex formation can occur between LH molecules originating from different species, and between different LH isoforms, double expressions were generated in a baculovirus system. Heterocomplex formation between mouse and human LH3, between human LH1 and LH3 and between human LH2 and LH3 was detected by western blot analyses. However, due to the low amount of complexes formed, the in vivo function of heterocomplexes remains unclear.