ARID1A loss induces P4HB to activate fibroblasts to support lung cancer cell growth, invasion, and chemoresistance.

Loss of AT-interacting domain-rich protein 1A (ARID1A) frequently occurs in human malignancies including lung cancer. The biological consequence of ARID1A mutation in lung cancer is not fully understood. This study was designed to determine the effect of ARID1A-depleted lung cancer cells on fibroblast activation. Conditioned media was collected from ARID1A-depleted lung cancer cells and employed to treat lung fibroblasts. The proliferation and migration of lung fibroblasts were investigated. The secretory genes were profiled in lung cancer cells upon ARID1A knockdown. Antibody-based neutralization was utilized to confirm their role in mediating the cross-talk between lung cancer cells and fibroblasts. NOD-SCID-IL2RgammaC-null (NSG) mice received tumor tissues from patients with ARID1A-mutated lung cancer to establish patient-derived xenograft (PDX) models. Notably, ARID1A-depleted lung cancer cells promoted the proliferation and migration of lung fibroblasts. Mechanistically, ARID1A depletion augmented the expression and secretion of prolyl 4-hydroxylase beta (P4HB) in lung cancer cells, which induced the activation of lung fibroblasts through the ß-catenin signaling pathway. P4HB-activated lung fibroblasts promoted the proliferation, invasion, and chemoresistance in lung cancer cells. Neutralizing P4HB hampered the tumor growth and increased cisplatin cytotoxic efficacy in two PDX models. Serum P4HB levels were higher in ARID1A-mutated lung cancer patients than in healthy controls. Moreover, increased serum levels of P4HB were significantly associated with lung cancer metastasis. Together, our work indicates a pivotal role for P4HB in orchestrating the cross-talk between ARID1A-mutated cancer cells and cancer-associated fibroblasts during lung cancer progression. P4HB may represent a promising target for improving lung cancer treatment.

MiR-204-5p regulates HUVEC cell inflammation and apoptosis by targeting P4HB.

The purpose of this study arose to investigate the mechanism of miR-204-5p targeting P4HB to regulate inflammation and apoptosis in HUVEC cells. Serum specimens were obtained from lower extremity DVT patients and healthy subjects. Targetscan predicted P4HB as a target gene for miR-204-5p. A dual luciferase reporter assay was conducted to determine the modulating effect of miR-204-5p on P4HB. qRT-PCR was used to detect miR-204-5p and P4HB expression. Established CoCl2-induced hypoxia/ischemia model of HUVEC, transfected with miR-204-5p mimics and pcDNA3. 1-P4HB. CCK-8 assay for cell viability. Apoptosis was assayed by flow cytometry, western blot and western blot. Immunofluorescence and ELISA were carried out to detect ROS, MDA, SOD, LDH, GSH-px, TNF-α, IL-1ß and IL-6 expression. miR-204-5p was reduced markedly in the sera of DVT patients. miR-204-5p negatively regulated P4HB. P4HB expression was raised in the sera of DVT patients. Exposure to CoCl2 decreased miR-204-5p expression and increased P4HB in HUVEC. Over-expressed miR-204-5p effectively increased cell viability and inhibited apoptosis; its effect was counteracted by continued overexpression of P4HB. In addition, miR- 204-5p mimics clearly reduced CoCl2-induced ROS and inflammation, and pcDNA3. 1-P4HB acted counteractively. miR-204-5p may inhibit HUVEC proliferation, ROS generation and cellular inflammation through negative regulation of P4HB. miR-204-5p promises to become a potential target for DVT therapy.

Prolyl 4-hydroxylase P4HA1 Mediates the Interplay Between Glucose Metabolism and Stemness in Pancreatic Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) are profoundly implicated in tumor initiation and progression as well as drug resistance and tumor recurrence of many cancer types, especially pancreatic ductal adenocarcinoma (PDAC). Previously, we revealed that prolyl 4-hydroxylase subunit alpha 1 (P4HA1) enhances the Warburg effect and tumor growth in PDAC. However, the possible connection between P4HA1 and cancer stemness in PDAC remains obscure. In this study, P4HA1-dependent cancer stemness was studied by sphere-formation assay and detection of stemness markers. METHODS: Glycolytic capacity in cancer stem cells and their parental tumor cells was investigated by glucose uptake, lactate secretion, and expression of glycolytic genes. Glycolysis inhibitors were used to determine the link between cancer stemness and glycolysis. A subcutaneous xenograft model was generated to investigate P4HA1-induced stemness and glycolysis in vivo. RESULTS: We revealed that ectopic expression of P4HA1 increased the stemness of PDAC cells as evidenced by the increased proportion of CD133+ cells, elevated sphere-formation ability, and the upregulated levels of cancer stemness-related proteins (SOX2, OCT4, and NANOG). Blocking tumor glycolysis with 2-Deoxy-D-glucose (2-DG) or a selective inhibitor of glucose transporter 1 (STF-31) significantly reduced the stem properties of PDAC cells, suggesting that P4HA1-induced glycolysis was essential for the stem-like phenotype of PDAC cells. In addition, in vivo study reaffirmed a promotive effect of P4HA1 on tumor glycolysis and cancer stemness. CONCLUSION: Collectively, our findings suggest that P4HA1 not only affects tumor metabolic reprogramming but also facilitates cancer stemness, which might be exploited as a vulnerable target for PDAC treatment.

Protein disulphide isomerase (PDI) is protective against amyotrophic lateral sclerosis (ALS)-related mutant Fused in Sarcoma (FUS) in in vitro models.

Mutations in Fused in Sarcoma (FUS) are present in familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS is localised in the nucleus where it has important functions in DNA repair. However, in ALS/FTD, mutant FUS mislocalises from the nucleus to the cytoplasm where it forms inclusions, a key pathological hallmark of neurodegeneration. Mutant FUS also inhibits protein import into the nucleus, resulting in defects in nucleocytoplasmic transport. Fragmentation of the neuronal Golgi apparatus, induction of endoplasmic reticulum (ER) stress, and inhibition of ER-Golgi trafficking are also associated with mutant FUS misfolding in ALS. Protein disulphide isomerase (PDI) is an ER chaperone previously shown to be protective against misfolding associated with mutant superoxide dismutase 1 (SOD1) and TAR DNA-binding protein-43 (TDP-43) in cellular and zebrafish models. However, a protective role against mutant FUS in ALS has not been previously described. In this study, we demonstrate that PDI is protective against mutant FUS. In neuronal cell line and primary cultures, PDI restores defects in nuclear import, prevents the formation of mutant FUS inclusions, inhibits Golgi fragmentation, ER stress, ER-Golgi transport defects, and apoptosis. These findings imply that PDI is a new therapeutic target in FUS-associated ALS.

Activation of the prolyl-hydroxylase oxygen-sensing signal cascade leads to AMPK activation in cardiomyocytes.

The proline hydroxylase domain-containing enzymes (PHD) act as cellular oxygen sensors and initiate a hypoxic signal cascade to induce a range of cellular responses to hypoxia especially in the aspect of energy and metabolic homeostasis regulation. AMP-activated protein kinase (AMPK) is recognized as a major energetic sensor and regulator of cardiac metabolism. However, the effect of PHD signal on AMPK has never been studied before. A PHD inhibitor (PHI), dimethyloxalylglycine and PHD2-specific RNA interference (RNAi) have been used to activate PHD signalling in neonatal rat cardiomyocytes. Both PHI and PHD2-RNAi activated AMPK pathway in cardiomyocytes effectively. In addition, the increased glucose uptake during normoxia and enhanced myocyte viability during hypoxia induced by PHI pretreatment were abrogated substantially upon AMPK inhibition with an adenoviral vector expressing a dominant negative mutant of AMPK-α1. Furthermore, chelation of intracellular Ca2+ by BAPTA, inhibition of calmodulin-dependent kinase kinase (CaMKK) with STO-609, or RNAi-mediated down-regulation of CaMKK α inhibited PHI-induced AMPK activation significantly. In contrast, down-regulation of LKB1 with adenoviruses expressing the dominant negative form did not affect PHI-induced AMPK activation. We establish for the first time that activation of PHD signal cascade can activate AMPK pathway mainly through a Ca(2+)/CaMKK-dependent mechanism in cardiomyocytes. Furthermore, activation of AMPK plays an essential role in hypoxic protective responses induced by PHI.

Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo.

Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.

Oxygen-dependent modulation of insulin-like growth factor binding protein biosynthesis in primary cultures of rat hepatocytes.

Higher levels of IGF-binding protein 1 (IGFBP-1) mRNA are expressed in the less aerobic perivenous zone of the liver. Because gradients in oxygen tension (pO(2)) may contribute to zonated gene expression, the influence of arterial and venous pO(2) on IGFBP-1 biosynthesis was studied in primary cultures of rat hepatocytes. Maximal IGFBP-1 mRNA and protein levels were observed under venous pO(2), whereas less than 30% of maximal levels were observed under arterial pO(2). In contrast, the expression of IGFBP-4 was greatest under arterial pO(2), indicating that this effect of hypoxia on IGFBP-1 gene expression is specific. The response to hypoxia appears to involve reactive oxygen species, because treatment with H(2)O(2) results in a dose-dependent decrease of IGFBP-1 mRNA levels under venous pO(2), whereas IGFBP-1 mRNA expression under arterial pO(2) was not affected. Inhibition of the hypoxia-dependent IGFBP-1 mRNA induction by actinomycin D indicates that this effect is mediated at the level of gene transcription, and inhibition of IGFBP-1 mRNA by the iron chelator desferrioxamine under both venous and arterial pO(2) suggested the involvement of hypoxia-inducible transcription factors (HIF). Transfection experiments demonstrated that especially HIF-3alpha and HIF-2alpha, and to a lesser extent HIF-1alpha, contribute to the induction of IGFBP-1 mRNA expression in isolated hepatocytes, whereas experiments with vectors for the HIF prolyl hydroxylases (PHD) indicated a major role of PHD-2 in destabilization of HIFs, attenuating the induction of IGFBP-1 under venous pO(2). Reporter gene studies indicate that hypoxia stimulates IGFBP-1 expression through a putative HIF response element located approximately 250 bp upstream from the transcription initiation site. Together, these results support the concept that iron, radical oxygen species, and the HIF-2 and -3 as well as the PHD pathways play important roles in mediating effects of hypoxia on IGFBP-1 gene expression in the liver.

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation.

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.

Prolyl 4-hydroxylase, a target enzyme for drug development. Design of suppressive agents and the in vitro effects of inhibitors and proinhibitors.

The hydrophilic compound pyridine 2,4-dicarboxylate (2,4-PDCA), designed as a mechanism-based competitive inhibitor of prolyl 4-hydroxylase, is efficiently excluded by the cytoplasmic membrane, but permeates the endoplasmic membrane via a 2,4-PDCA-selective translocator to reach its target enzyme in the intracisternal space. A lipophilic 2,4-PDCA-based proinhibitor, inactive with purified prolyl 4-hydroxylase, shows a cell system-dependent suppression of hydroxyprolyl formation, displaying a half-maximally inhibitory concentration very similar to the Ki of the parent compound. Apparently, cell-specific intracellular metabolic processing of the proinhibitor regenerates the active agent, 2,4-PDCA. The in vitro findings summarized here suggest that the 2,4-PDCA-mediated inhibition of prolyl 4-hydroxylase has a marked disruptive effect on the biosynthesis and deposition of collagen. This effect qualifies 2,4-PDCA and its derivatives as experimental fibrosuppressive compounds. However, to avoid catastrophic consequences in vivo, it is desirable to target the active agent to only the tissue that is compromised by excessive matrix formation. This requirement can be realized by the deliberate selection of an appropriate, 2,4-PDCA-based proinhibitor and by the deliberate selection of the route of proinhibitor administration.

Effects of joint loading on articular cartilage collagen metabolism: assay of procollagen prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase.

The degree of joint loading on the femoral head cartilage was experimentally altered in dogs by splinting of the knee joint for 11 weeks (n = 9), and making another group (n = 6) to run on a treadmill with 15 degrees uphill inclination during a period of 15 weeks (4 km/day, 5 days a week). A third group (n = 9) served as controls. The influence of these altered loading conditions on articular cartilage collagen synthesis was measured by assaying the activity of procollagen prolyl 4-hydroxylase (PPH) and galactosylhydroxylysyl glucosyltransferase (GGT) in chondrocytes. The average activity of PPH was 11-13% elevated in the runner and contralateral (more loaded) cartilages, while the splinted (unloaded) cartilages showed a significant (53%) increase of PPH. In some samples of the runner, splinted and contralateral cartilages the activity of GGT was also high, but the differences did not reach statistical significance. The increase of the activity of PPH, combined with unaltered total content of collagen, indicates that the synthesis of collagen and probably also its turnover, are enhanced in the cartilage atrophied due to reduced weight-bearing, but only slightly, if at all in the cartilages subjected to moderately elevated loading.

The relationship between collagen and C1q biosynthesis in cultured human fibroblasts.

The relationship between collagen and C1q biosynthesis has been investigated in cultured human fibroblasts. This was done by measuring the effects of variation in cell density, inhibition of prolyl hydroxylase and complexing of C1q on synthesis and/or secretion of both proteins. It was found that synthesis and secretion of both proteins were not co-ordinate, and that therefore regulation of expression of both proteins is probably not linked. However complexing of C1q did result in marked stimulation in collagen synthesis, suggesting that the fibrosis which follows inflammation may result from binding of C1q to the immune complexes formed in the inflammatory process.

Cortisol effect on collagen biosynthesis in embryonic explants and in vitro hydroxylation of protocollagen.

The effect of increasing doses of hydrocortisone acetate, hydrocortisone phosphoric acid complex, and hydrocortisone sodium succinate on collagen biosynthesis was assayed in two different systems. I. Explants of chicken embryo tibiae showed decreased biosynthesis of [14C]hydroxyproline and total and glycosylated [14C]hydroxylysine under the influence of high doses of hydrocortisone. With the lower doses of hydrocortisone acetate, no effect was noticed. The total uptake of the precursor amino acids followed patterns similar to those of collagen biosynthesis. II. Hydroxylation of [14C]proline labelled protocollagen by protocollagen proline hydroxylase was inhibited by high doses of hydrocortisone acetate, hydrocortisone phosphoric acid complex, and hydrocortisone sodium succinate. III. A decreased diffusion of collagen to the medium with increasing doses of hydrocortisone acetate, hydrocortisone phosphoric acid, and hydrocortisone sodium succinate was noticed. IV. No further hydroxylation of new-synthesized collagen was obtained under the influence of hydrocortisone phosphoric acid and hydrocortisone sodium succinate, when the undialyzable material was used as a substrate for protocollagen proline hydroxylase.