Precede-Proceed Model-dominant Nursing Combined With Nutrition Support for Patients Discharged After Hepatobiliary Surgery With T Tube.

Context: Hepatobiliary disease requires surgical treatment and T-tube installment postoperatively, and discharged patients' usually still have a T tube. Little nursing care is available in China for patients after discharge, resulting in postdischarge complications. Also, the incidence of nutritional risk in patients with hepatobiliary surgery is high. Objective: The study aimed to investigate the benefits of a precede-proceed model-dominant nursing combined with nutritional support for patients discharged after hepatobiliary surgery with a T tube, so as to improve their prognoses and promote their rehabilitation. Design: The research team conducted a prospective, single-center, randomized controlled trial. Setting: The study took place at Wuhan No. 1 Hospital, Wuhan Hospital of Traditional Chinese and Western Medicine in Wuhan, Hubei, China. Participants: Participants were 120 patients discharged after hepatobiliary surgery at the hospital between June 2020 and June 2022. Interventions: The research team randomly divided participants into two groups using the random number table method, each with 60 participants: (1) an intervention group, which received precede-proceed model-dominant nursing combined with nutrition support and (2) a control group, which received routine care. Outcome Measures: At baseline and postintervention, the research team assessed: (1) nutritional status, (2) self-care agency, (3) compliance, (4) quality of life (QoL), (5) incidence of complications. Results: At baseline, no significant differences existed between the groups in nutritional status, self-care agency, QoL, or compliance (all P > .05). Postintervention compared to the control group, the intervention group's: (1) nutritional status, including albumin (P = .015), hemoglobin (P < .001), growth hormone (P < .001), BW (P = .047), BMI (P = .046), TST (P = .001), and MAMC (P = .016) were significantly higher and transferrin (P < .001) and NRS-2002 score (P < .001) were significantly lower; (2) self-care agency, including self-concept, self-responsibility, health knowledge, and self-nursing skills were significantly higher (all P < .001); (3) compliance scores, including observing the volume and color of bile correctly, clamping and opening the T tube properly, replacing the drainage bag correctly and in a timely manner, regularly disinfecting the skin around the drainage tube, keeping a balanced diet, adhering to medical regimens, exercising adequately were significantly higher (all P < .001); (4) QoL was significantly higher (P < .001); and (5) incidence of complications was significantly lower (P = .008). Conclusions: Precede-proceed model-dominant nursing combined with nutrition support can significantly improve nutritional status, self-care agency, and QoL and can significantly decrease the incidence of complications for patients discharged after hepatobiliary surgery with a T tube and is worthy of promotion in clinics.

Effect of lncRNA XIST on acute myeloid leukemia cells via miR-142-5p-PFKP axis.

Acute myeloid leukemia (AML) is the common blood cancer in hematopoietic system-related diseases and has a poor prognosis. Studies have shown that long non-coding RNAs (lncRNAs) are closely related to the pathogenesis of a variety of diseases, including AML. However, the specific molecular mechanism remains unclear. Hence, the objective of this study was to investigate the effect and mechanism of lncRNA X inactive specific transcript (lncRNA XIST) on AML. To achieve our objective, some tests were performed. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of lncRNA XIST, miR-142-5p and the platelet isoform of phosphofructokinase (PFKP). The targeting relationship between miR-142-5p and lncRNA XIST and PFKP was verified by Pearson correlation analysis, dual-luciferase reporter assay, and pull-down assay. Functional experiments were used to analyze the effect and mechanism of action of knocking down lncRNA XIST on THP-1 and U937 cells. Compared with bone marrow cells, lncRNA XIST and PFKP expression levels were up-regulated and miR-142-5p expression levels were down-regulated in AML. Further analysis revealed that lncRNA XIST targeted and bound to miR-142-5p, and PFKP was a target gene of miR-142-5p. Knockdown of lncRNA XIST significantly promoted miR-142-5p expression to down-regulate PFKP in THP-1 and U937 cells, while the cell proliferation, cell viability, and cell cycle arrest were inhibited and apoptosis was increased. Knockdown of miR-142-5p reversed the functional impact of lncRNA XIST knockdown on AML cells. In conclusion, down-regulation of lncRNA XIST can affect the progression of AML by regulating miR-142-5p.

Effect of accelerated rehabilitation combined with enteral nutrition on gastrointestinal function recovery after hepatectomy.

OBJECTIVE: To investigate the effect of accelerated rehabilitation combined with enteral nutrition on the recovery of gastrointestinal function in patients after hepatectomy. METHODS: In this prospective randomized controlled study, a total of 108 patients with liver cancer who underwent hepatectomy in our hospital from November 2020 to December 2021 were selected as the research subjects. According to the random number table method, the patients were divided into a research group (54 cases, who received accelerated rehabilitation combined with enteral nutrition) and a control group (54 cases, who received routine nursing without enteral nutrition). The gastrointestinal function of patients in two groups was recorded and compared, the nutritional status before and after surgery was compared, and the levels of T cell subsets CD3+, CD4+, CD8+, CD4+/CD8+, and liver function were compared between the two groups. The adverse reactions such as catheter infection, diarrhea, vomiting, intestinal obstruction, and ascites occurred in two groups were recorded. RESULTS: The gastrointestinal function of patients in research group was significantly better than that in control group (P < 0.05). After intervention, the albumin (ALB) in research group was significantly higher than that in control group (P < 0.05), but no significant difference in body weight between two groups was found (P > 0.05). After intervention, CD3+, CD4+, CD8+, and CD4+/CD8+ in the research group were significantly higher than those in the control group (P < 0.05). In addition, there was no significant difference in liver function indexes between the two groups (P > 0.05); however, the incidence of adverse reactions in research group was significantly lower than that in the control group (P < 0.05). CONCLUSION: Accelerated rehabilitation combined with enteral nutrition can effectively restore the gastrointestinal function of patients after hepatectomy, which is worthy of clinical promotion.

LINC-PINT impedes DNA repair and enhances radiotherapeutic response by targeting DNA-PKcs in nasopharyngeal cancer.

Radioresistance continues to be the leading cause of recurrence and metastasis in nasopharyngeal cancer. Long noncoding RNAs are emerging as regulators of DNA damage and radioresistance. LINC-PINT was originally identified as a tumor suppressor in various cancers. In this study, LINC-PINT was significantly downregulated in nasopharyngeal cancer tissues than in rhinitis tissues, and low LINC-PINT expressions showed poorer prognosis in patients who received radiotherapy. We further identified a functional role of LINC-PINT in inhibiting the malignant phenotypes and sensitizing cancer cells to irradiation in vitro and in vivo. Mechanistically, LINC-PINT was responsive to DNA damage, inhibiting DNA damage repair through ATM/ATR-Chk1/Chk2 signaling pathways. Moreover, LINC-PINT increased radiosensitivity by interacting with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and negatively regulated the expression and recruitment of DNA-PKcs. Therefore, these findings collectively support the possibility that LINC-PINT serves as an attractive target to overcome radioresistance in NPC.

LncRNA linc00312 suppresses radiotherapy resistance by targeting DNA-PKcs and impairing DNA damage repair in nasopharyngeal carcinoma.

Radioresistance is the main obstacle in the clinical management of nasopharyngeal carcinoma (NPC). linc00312 is deregulated in a number of human cancers, including NPC. However, the detailed functions and underlying mechanisms of linc00312 in regulating radiosensitivity of NPC remains unknown. In this study, cox regression analysis was used to assess the association between linc00312 and NPC patients' survival after radiotherapy. Our results reveal that linc00312 is significantly down-regulated in NPC tissues and patients with higher expression of linc00312 are significantly associated with longer overall survival and better short-term radiotherapy efficacy. Overexpression of linc00312 could increase the sensitivity of NPC cells to ionizing radiation, as indicated by clonogenic survival assay, comet assay, and flow cytometry. Mechanistically, RNA pull down and RNA immunoprecipitation were performed to investigate the binding proteins of linc00312. linc00312 directly binds to DNA-PKcs, hinders the recruitment of DNA-PKcs to Ku80, and inhibits phosphorylation of AKT-DNA-PKcs axis, therefore inhibiting the DNA damage signal sensation and transduction in the NHEJ repair pathway. In addition, linc00312 impairs DNA repair and cell cycle control by suppressing MRN-ATM-CHK2 signal and ATR-CHK1 signal. In summary, we identified DNA-PKcs as the binding protein of linc00312 and revealed a novel mechanism of linc00312 in the DNA damage response, providing evidence for a potential therapeutic strategy in NPC.

Efficacy and Safety of Image-Guided Intensity-Modulated Radiation Therapy and Volumetric Modulated Arc Therapy Combined with Paclitaxel Liposomes and Cisplatin for Locally Advanced Stage IIB-IIIB Cervical Cancer: A Retrospective Study at a Single Center.

BACKGROUND This retrospective study aimed to investigate the efficacy and safety of image-guided intensity-modulated radiation therapy (IMRT) and volumetric modulated arc therapy (VMAT) combined with administration of paclitaxel liposomes and cisplatin for locally advanced stage IIB-IIIB cervical cancer at a single center in China. MATERIAL AND METHODS The clinical data of 126 patients with stage IIB-IIIB cervical cancer treated in our hospital were retrospectively analyzed. The patients were divided into the IMRT group (n=63) and the VMAT group (n=63). The short-term clinical efficacy, the incidence of adverse reactions, the quality-of-life score, and the changes in levels of T-lymphocyte subsets, serum inflammatory factors, and tumor markers were compared pre- and posttreatment between the 2 groups. RESULTS The clinical response rate was 90.5% and 96.8% in the IMRT group and the VMAT group, respectively; the difference was not statistically significant. After treatment, the levels of CD3⁺, CD4⁺, and CD4⁺/CD8⁺ subsets rose significantly, while the CD8⁺ level declined significantly in both groups compared with the pretreatment levels. After treatment, the levels of serum vascular endothelial growth factor, squamous cell carcinoma antigen, interleukin-8, tumor necrosis factor-a, carcinoembryonic antigen, and carbohydrate antigen 125 declined in both groups compared with pretreatment levels. After treatment, the Karnofsky performance scale score rose in both groups, and it was higher in the VMAT group than in the IMRT group. CONCLUSIONS IMRT and VMAT combined with paclitaxel liposomes and cisplatin have similar short-term clinical efficacy and long-term survival rates in the treatment of stage IIB-IIIB cervical cancer.

A functional variant in CHK1 contributes to increased risk of nasopharyngeal carcinoma in a Han Chinese population.

DNA damage checkpoints act as a supervisor by preventing the course of cell cycle upon DNA damage and keeping the steadiness of genome. Checkpoint kinase 1 (CHK1) cannot be ignore in the etiology of numerous human cancers including nasopharyngeal cancer (NPC). To discuss genetic polymorphisms of CHK1 rs492510 in the occurrence of NPC was our objective. Rs492510 polymorphism of CHK1 was genotyped in 684 patients with NPC and 823 cancer-free controls. We utilize logistic regression models to appraise the correlation of rs492510 and susceptibility of NPC. Comparative expression level about CHK1 in nasopharyngeal carcinoma tissues were determined by real-time polymerase chain reaction. And we made use of Dual-Luciferase Reporter Assay to assess the transcriptional ability of CHK1 with different rs492510 allele. Adjusting multivariate logistic regression based on age, sex, body mass index, smoking, and drinking status showed that CHK1 rs492510 GA + GG genotype carriers presented prominent higher risk in NPC (odds ratio = 1.376, 95% confidence interval: 1.087-1.742; P = .008). As a consequence, we revealed that CHK1 relative expression levels in NPC tissues was higher than rhinitis tissues. Besides, the expressions of CHK1 in rs492510 GA genotype carriers were higher compared with people in AA genotype. The G allele of rs492510 generated remarkable higher transcription activity of CHK1 vs A allele by luciferase reporter assay. Our study considered that single nucleotide polymorphism rs492510 could increase transcription activity of CHK1 with the functionality, contributing to the susceptibility of NPC.

miR-3188 (rs7247237-C>T) Single-Nucleotide Polymorphism Is Associated With the Incidence of Vascular Complications in Chinese Patients With Type 2 Diabetes.

miR-3188, one of the earliest discovered microRNAs, is involved in regulating the mTOR-p-PI3K/AKT pathway, thus affecting the progression of diabetic complications. In this study, we observed that the miR-3188 (rs7247237-C>T) polymorphism not only affected the production of nitric oxide (NO) production in endothelial cells, but also significantly associated with the incidence of vascular complications in Chinese patients with type 2 diabetes. Mechanistic analyses indicate that miR-3188 (rs7247237-T) polymorphism inhibited its own expression and upregulated the expression of gstm1 and trib3, which impairs NO production in human endothelial cells through inactivating AKT/eNOS signal transduction pathway. In addition, our clinical retrospective study indicated that, compared with patients with the CC genotype (n = 351), patients with rs7247237 TT + CT genotypes (n = 580) exhibited an increased risk of major vascular events during intensive glucose control treatment (hazard ratio = 1.560; 95% CI: 1.055-2.307, P = 0.025). Simultaneously, the risk of major vascular events was marginally decreased in patients with the CC genotype during intensive glucose control treatment compared with standard treatment (hazard ratio = 0.666; 95% CI: 0.433-1.016, P = 0.053). Our findings indicate that the miR-3188 (rs7247237-C>T) polymorphism is associated with the incidence of vascular complications in Chinese patients with type 2 diabetes, likely due to its remarkable effect on miR-3188 expression.

A Functional 5'-UTR Polymorphism of MYC Contributes to Nasopharyngeal Carcinoma Susceptibility and Chemoradiotherapy Induced Toxicities.

MYC is a transcription factor acting as a pivotal regulator of genes involved in cell cycle progression, apoptosis, differentiation and metabolism. In this study, we evaluated the association of MYC polymorphisms with nasopharyngeal carcinoma (NPC) risk and chemoradiotherapy induced toxicities among Chinese population. By using bioinformatic tools, five potential functional single nucleotide polymorphisms of MYC were genotyped in a case-control study with 684 NPC patients and 823 healthy controls. We found two SNPs rs4645948 (C>T) and rs2071346 (G>T) were significantly associated with increased risk of developing NPC (TT+CT vs CC, OR=1.557, P=3.34×10-4; TT+GT vs GG, OR=1.361, P=0.007, respectively). In addition, rs4645948 (C>T) was conferred with increased risk of anemia (CT vs CC, OR=2.152, P=0.001) and severe leukopenia (CT vs CC, OR=1.893, P=0.034) for NPC patients receiving chemoradiotherapy. We also found rs2071346 (G>T) variant genotype carriers were subjected to higher risk of anemia (GT vs GG, OR=1.665, P=0.022) and thrombocytopenia (GT vs GG, OR=1.685, P=0.035). Our results demonstrated that the relative expression of MYC was dramatically higher in NPC tissues compared to rhinitis tissues. Over-expression of MYC was positively correlated with advanced T stage, N stage, and late clinical stage. Notably, the expression of MYC in rs4645948 CT and TT genotypes carriers were significantly higher than CC genotype carriers. Luciferase reporter assay indicated that the T allele of rs4645948 led to significantly higher transcription activity of MYC compared to the C allele. These findings suggested that individual carrying the rs4645948 T allele may be at greater risk for NPC due to an increase of MYC transcriptional activity and an augment of MYC expression.

The pivotal oncogenic role of Jab1/CSN5 and its therapeutic implications in human cancer.

Jab1/CSN5 is a conserved multifunctional protein involved in ubiquitin-mediated protein degradation. Deregulation of Jab1/CSN5 can exert dramatic effects on diverse cellular functions, including DNA repair, cell cycle control, apoptosis, angiogenesis, and signal transduction, all of which are critical for tumor development. Although increasing evidence has demonstrated that Jab1/CSN5 was overexpressed in a variety of human cancers and usually correlated with poor prognosis, little was known about the underlying regulatory principles that coordinated its function. In this review, we highlight recent advances of the oncogenic role of Jab1/CSN5 and its potential as a therapeutic target for anticancer intervention.

Author Correction: Genetic polymorphisms of lncRNA-p53 regulatory network genes are associated with concurrent chemoradiotherapy toxicities and efficacy in nasopharyngeal carcinoma patients.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Genetic polymorphisms of long non-coding RNA GAS5 predict platinum-based concurrent chemoradiotherapy response in nasopharyngeal carcinoma patients.

LncRNA GAS5 plays a tumor suppressive role in a variety of human cancers and promises to be a novel diagnostic biomarker, therapy target, as well as prognostic biomarker. However, the role of GAS5 in nasopharyngeal carcinoma (NPC) remains elusive. The objective of the present study was to evaluate the effect of single nucleotide polymorphisms (SNPs) in GAS5 on treatment efficacy and toxicity in NPC patients receiving chemoradiotherapy. Three potentially functional SNPs of GAS5 were genotyped in 267 NPC patients and validated in another 238 NPC patients treated with chemoradiotherapy from southern China. Multivariate logistic regression analyses and stratification analyses were used to estimate the association of candidate SNPs and chemoradiotherapy efficacy and toxic reactions. Our results showed that rs2067079 kept a consistent association with severe myelosuppression and severe neutropenia in discovery set (OR=2.403, P=0.009; OR=2.454, P=0.015; respectively), validation set (OR=3.653, P=0.027; OR=4.767, P=0.016; respectively), and combined dataset (OR=1.880, P=0.007; OR=2.079, P=0.005; respectively). rs2067079 CT genotype carriers presented an even more remarkable increased risk of severe myelosuppression (OR=3.878, P=0.003) and severe neutropenia (OR=3.794, P=0.009) in subgroups taking paclitaxel+platinum as concurrent chemoradiotherapy regimen. Besides, we found a gene-does effect of rs6790, with the incidence rate of severe myelosuppression decreased from 23.56% to 17.21% to 10% and the incidence rate of severe neutropenia decreased from 30.4% to 20.9% to 17.1% for rs6790 GG vs GA vs AA genotype carriers. Our results indicate the potential role of lncRNA GAS5 polymorphisms rs2067079 and rs6790 as predictive biomarkers for chemoradiotherapy induced toxic reactions in NPC patients.

Genetic polymorphisms of lncRNA-p53 regulatory network genes are associated with concurrent chemoradiotherapy toxicities and efficacy in nasopharyngeal carcinoma patients.

The relevance of the transcription factor p53 in cancer is inarguable, and numerous lncRNAs are involved in the p53 regulatory network as either regulators or effectors, triggering a transcriptional response that causes either cell arrest or apoptosis following DNA damage in a p53-dependent manner. Despite the fact that the therapeutic response is improved in NPC, heterogeneity among people remains with regard to the susceptibility of adverse effects and the efficacy of treatments. Therefore, we analysed eight potentially functional SNPs of five genes in the lncRNA-p53 regulatory network in a discovery cohort of 505 NPC patients. By performing multivariate logistic regression, the impact of genetic variations on the efficacy and risk of CRT-induced toxicities was investigated. The most dramatic finding was that the MEG3 rs10132552 CC genotype had a greater than three-fold increased risk of developing grade 3-4 anaemia (OR = 3.001, 95%CI = 1.355-6.646, P = 0.007). Furthermore, the rs10132552 CT genotype had a better response to treatment (OR = 0.261, 95%CI = 0.089-0.770, P = 0.015). Individuals carrying LINC-ROR rs2027701 with one or two variant alleles had significant associations with a reduced risk of neutropaenia (OR = 0.503, 95%CI = 0.303-0.835, P = 0.008). In conclusion, our results suggested that genetic polymorphisms of the lncRNA-p53 regulatory network could play a potential role in reducing treatment-related toxicities and improving outcomes for NPC patients.

BART miRNAs: an unimaginable force in the development of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a head and neck cancer that represents a major health burden in Southern China and Southeast Asia. Although the close association of NPC with Epstein-Barr virus (EBV) infection has been demonstrated, its exact role in the pathogenesis of this malignancy is still unclear. The expression of EBV-encoded microRNAs, especially BART miRNAs, which are encoded from the BamHI-A region of the viral genome, is detected at a high level in NPC. miRNAs are small noncoding mRNAs that can positively regulate the virus to ensure accurate expression of viral genomes and to modify the gene expression of host cells by negative regulation. Accumulating evidence suggests that ebv-mir-BARTs play a critical role in host cell survival, immune escape, cell proliferation, cell apoptosis, and cancer metabolism, promoting the generation of NPC. This review will summarize our current understanding of the nature and function of ebv-mir-BARTs in NPC.

ELMO1 Directly Interacts with Gßγ Subunit to Transduce GPCR Signaling to Rac1 Activation in Chemotaxis.

Diverse chemokines bind to G protein-coupled receptors (GPCRs) to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. ELMO and Dock proteins form complexes that function as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR activation and the ELMO/Dock-mediated Rac activation is not fully understood. In the present study, we show that chemoattractants induce dynamic membrane translocation of ELMO1 in mammalian cells. ELMO1 plays an important role in GPCR-mediated chemotaxis. We also reveal that ELMO1 and Dock1 form a stable complex. Importantly, activation of chemokine GPCR promotes the interaction between ELMO1 and Gßγ. The ELMO1-Gßγ interaction is through the N-terminus of ELMO1 protein and is important for the membrane translocation of ELMO1. ELMO1 is required for Rac1 activation upon chemoattractant stimulation. Our results suggest that chemokine GPCR-mediated interaction between Gßγ and ELMO1/Dock1 complex might serve as an evolutionarily conserved mechanism for Rac activation to regulate actin cytoskeleton for chemotaxis of human cells.

Aging increases the susceptibility of cisplatin-induced nephrotoxicity.

Cisplatin (CDDP) nephrotoxicity is one of the most common side effects in cancer treatment, causing the disruption of chemotherapy. In this study, we analyzed the influence of nongenetic factors on CDDP-induced nephrotoxiciy using the data from 182 CDDP-treated and 52 carboplatin (CBP)-treated patients. The mean change of eGFR (100% to baseline) in CDDP-treated patients was -9.2%, which was significantly lower than that in the population with CBP therapy. By using the chi-squared test and multivariate logistic regression analysis, age (≥50 years) is found associated with CDDP-induced nephrotoxicity, with odds ratio (OR) of 9.167 and 11.771, respectively. Three- and 18-month-old mice were employed to study the age-dependent susceptibility of CDDP-induced nephrotoxicity. Biochemical parameters, histopathogical examination, and mRNA biomarkers indicated that old mice were subjected to more severe kidney injury. In addition, old mice accumulated more CDDP in kidney than young mice, and the protein level of CDDP efflux transporter, MATE1, in aged mice kidney was 35% of that in young mice. Moreover, inflammatory receptor TLR4 was higher in the kidney of old mice, indicating the alteration of inflammatory signaling in old mice. After CDDP administration, the induced alterations of TNF-α, ICAM-1, and TLR4 were more extensive in old mice. To summarize, aging increased the susceptibility of CDDP-induced renal function decline or nephrotoxicity.

2-(N-Alkylcarboxamide)-6-iminopyridyl palladium and nickel complexes: coordination chemistry and catalysis.

The 2-(N-alkylcarboxamide)-6-iminopyridine ligands (L1-L7) can bind as either mono-anionic tridentate N^N^N ligands on reaction with PdCl(2)(CH(3)CN)(2), to form complexes LPdCl (C1-C7), or as neutral tridentate N^N^O ligands with NiCl(2)·6H(2)O, to produce complexes LNiCl(2) (C8-C14). All metal complexes were characterized by IR spectroscopy and elemental analysis, and in the case of the palladium complexes, by (1)H and (13)C NMR spectroscopy. The crystal structures of C3, C4, C6, C10, and C12 were determined by X-ray crystallography, and revealed a distorted square geometry around the palladium centre, whereas for nickel, a distorted square-pyramidal geometry was adopted. The representative palladium complex (C3) was further reacted with AgBF(4) in acetonitrile affording the salt [L3Pd(CH(3)CN)][BF(4)] (C15) and the structure of this was confirmed by single-crystal X-ray diffraction. By contrast, carrying out the reaction in dichloromethane rather than acetonitrile, in the presence of malononitrile (CNCH(2)CN), resulted in the formation of the bimetallic palladium complex [L3Pd(CNCH(2)CN)PdL3]·2[BF(4)] (C16). Upon activation with diethylaluminium chloride, all the nickel complexes showed high activity for ethylene dimerization. Furthermore, the palladium complexes exhibited good activities in the vinyl-polymerization of norbornene upon activation with MAO.

Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex.

We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -ß) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.

Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain.

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 microg/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.