[Clinical study of percutaneous vertebroplasty combined with (125)I seeds implantation in the treatment of patients with thoracic metastatic tumor complicated with posterior vertebral defect].

Objective: To analyze the safety and efficacy of percutaneous vertebroplasty combined with interstitial implantation (125)I of seeds (PVPI) in the treatment of thoracic vertebroplasty with posterior vertebra defect. Methods: A retrospective analysis of the clinical data of 64 patients with thoracic spine metastases admitted to Yunnan Cancer Hospital from November 2017 to May 2019 was conducted, including 32 patients with posterior vertebra defect (experimental group) and 32 cases without (control group). Forty-two vertebral bodies of 32 patients in the experimental group were treated with improved PVPI surgery, which performed with the secondary sealing method and inclined puncture needle injection bone cement rotary filling technology, to reduce leakage. The 54 vertebral bodies of 32 patients in control group underwent PVPI. The two groups of patients were followed up on the second day, one month, three months and six months after the operation, and the short-term efficacy, long-term efficacy and safety indicators of the two groups were compared. Results: All 64 patients successfully completed the surgical treatment. The visual analogue scores and Karnofsky scores of the experimental group and the control group were improved to varying degrees on the second day, 1 month, 3 months and 6 months after the operation. There was no statistically significant difference between the two groups (P>0.05). The amount of bone cement in the experimental group and control group was (2.36±0.20) ml and (2.39±0.17) ml, and the difference was not statistically significant (P=0.482). The amount of (125)I seed implantation was (30.63±0.91) and (32.56±0.68), respectively, the difference was not statistically significant (P=0.925). The partial response rates of the study group and the control group were 81.3% and 87.5%, the stable disease rates were 12.5% and 9.4%, the differences were not statistically significant (P>0.05). The median overall survival (mOS) of the study group was 13 months, and the median progression-free survival (mPFS) was 8 months. The mOS of the control group was 14 months, and the mPFS was 8 months. The differences were not statistically significant (P>0.05). In the experimental group, 6 (14.3%) vertebral bodies had cement leakage, of which 2 (4.8%) were cement leakage at posterior vertebra, 4 (9.5%) were paravertebral cement leakage. Seven (13.0%) paravertebral cement leakage occurred in the control group. There was no significant difference in bone cement leakage between the two groups (P=0.097). Bone cement leakage in both groups did not cause serious complications such as spinal cord injury and paraplegia. Conclusion: The application of PVPI in the treatment of thoracic metastatic tumor patients with posterior vertebra defect can acquire better clinical efficacy and safety through conduction of the improved intraoperative technology and paying more attention to the control of bone cement distribution and other issues.

[Application of robot-assisted surgery in the surgical treatment of head and neck cancer].

In recent years, robot-assisted surgery (RAS) has developed rapidly and become one of the hot topics in clinical research. Compared with traditional surgery, RAS has advantages in terms of minimal invasiveness, aesthetics, and functional preservation, and has been gradually applied in clinical practice such as neurosurgery, urology, and head and neck surgery. In the treatment of head and neck tumors, RAS can effectively minimize the surgical injury and accelerate postoperative recovery. This article reviews the application of RAS in the resection of primary lesions of head and neck tumors, neck dissection, and reconstruction of tissue defects.

[Clinical significance of syndecan-1 and syndecan-2 expression in gallbladder squamous cell/adenosquamous carcinoma and adenocarcinoma].

Objective: To investigate the expression of syndecan-1 and syndecan-2 and their clinicopathological significance in patients with gallbladder squamous cell (SC)/adenosquamous carcinoma (ASC) and adenocarcinoma (AC). Methods: A total of 126 patients with SC/ASC (n=46) and AC (n=80) were included in this study. The expression levels of syndecan-1 and syndecan-2 were detected by Envison™ immunohistochemistry assay. The clinical and prognostic significance of syndecan-1 and syndecan-2 were analyzed. Results: In the 46 SC/ASC samples, syndecan-1 and syndecan-2 were positively expressed in 29 (63.0%) and 28 (60.9%) tumor tissues, respectively. (Positive expression was defined based on the staining in the component of squamous cell carcinoma. That is to say, the tissue which adenocarcinoma part was positively stained, but squamous cell carcinoma part was negatively stained is also regarded as negative.) In the 80 AC samples, 47 (58.8%) cases showed syndecan-1 positive expression, and 51 (63.8%) showed syndecan-2 positive expression. There was no significant difference in the positive rates of syndecan-1 and syndecan-2 between SC/ASC and AC groups (P>0.05 for all). The levels of syndecan-1 and syndecan-2 were associated with tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in SC/ASC patients (P<0.05 for all). However, their expression was associated with tumor differentiation, tumor size, TNM staging, lymph node metastasis, invasion of adjacent tissue, and surgical procedures in AC patients (P<0.05 for all). The Kaplan-Meier survival analysis of SC/ASC and AC patients revealed that the average survival time for patients with positive syndecan-1 and syndecan-2 expression was significantly shorter than that of those with negative expression (P<0.01 for all). Cox multivariate analysis indicated that syndecan-1 and syndecan-2 expression were independent unfavorable prognostic factors for SC/ASC and AC patients (P<0.05 for all). Conclusion: The syndecan-1 and syndecan-2 expression are associated with the tumor progression and poor prognosis in patients with gallbladder SC/ASC and AC.

Physiological dissection revealed that both uptake and assimilation are the major components regulating different growth responses of two tobacco cultivars to nitrogen nutrition.

K326 and HD represent major tobacco cultivars in China, which required large N fertiliser input but at different application rates. To understand primary components affecting tobacco N use physiology, we adopted these two varieties as valuable genetic material to assess their growth response to N nutrition. We established a hydroponic culture system to grow plants supplied with different N regimes. Plant biomass, N, ammonium, nitrate, arginine, GS and NR activity, N transfer and use efficiency as well as root uptake were examined. Our data revealed the preference of K326 and HD to utilise nitrate or ammonium nitrate but not ammonium alone, with 2 mm N supply probably sufficient and economical to achieve good biomass production at the vegetative stage. Moreover, both varieties were very sensitive to ammonium, perhaps due to lack of or abnormal signalling related to nitrate and/or arginine rather than impairment of N acquisition and initial assimilation; this was supported by measurements of the plant content of N, ammonium and activities of GS and NR. Notably, short-term 15 N root influx studies identified differential uptake kinetics of K326 and HD, with distinct affinities and transport rates for ammonium and nitrate. The data suggest that the growth adaptation of K326 or HD to higher or lower N may be ascribed to different competences for effective N uptake/translocation and assimilation. Thus, our work provides valuable information to prompt deeper investigation of the molecular basis controlling plant N use efficiency.

shRNA-mediated silencing of hTERT suppresses proliferation and promotes apoptosis in osteosarcoma cells.

In this study, we aim to explore the effects of short hairpin RNAs (shRNAs) targeting human telomerase reverse transcriptase (hTERT) on the proliferation and apoptosis of osteosarcoma cells. After the synthesis of shRNA that target hTERT, osteosarcoma cells were assigned into three experimental groups-shRNA group, scramble group and blank group. The transcription and expressions of the hTERT gene in transfected cells were measured with quantitative real-time polymerase chain reaction and western blotting. Cell proliferation in each group was detected by Cell Counting Kit-8 assay. Cell cycle and rates of apoptosis were measured by flow cytometry. Expressions of apoptosis-related proteins, caspase-9 and caspase-3, were detected by western blotting. Telomerase activity was measured by PCR enzyme-linked immunosorbent assay. Results show that both the mRNA and protein expressions of hTERT were significantly lowered after the transfection of hTERT-shRNA. The proliferation capacity of transfected osteosarcoma MG-63, SaOS2 and U2OS cells in the shRNA group was lower than that in the blank group. We also found changes and differences in the amount of cells throughout the cell cycle. All cells in the G0/G1 phase increased in numbers, whereas the number of cells in the S phase were reduced, with elevated apoptosis rates. Expressions of apoptosis-related proteins, caspase-9 and caspase-3, were increased and telomerase activity was decreased in the transfected shRNA group (all P<0.05). Our results showed that shRNA targeting of the hTERT gene was able to inhibit cell proliferation and promote apoptosis of osteosarcoma cells by reducing the telomerase activity.

[Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1].

Objective: To investigate the killing effects of radiation and mutant Rad50 transfection on human nasopharyngeal carcinoma cell line CNE1. Methods: The experimental groups included: control group, Ad-Rad50-GFP group, Ad-EGFP group, irradiation group, Ad-Rad50-GFP combined with irradiation group, and Ad-EGFP combined with irradiation group. CNE1 cells were transfected with recombinant adenoviral vector Ad-Rad50-GFP carrying mutant Rad50 gene. The expressions of Mre11, Rad50, Nbs1, and relevant constituents composing MRN complex were detected by Western Blot. Neutral comet assay was used to detect the effect of mutant Rad50 on restoration process of DNA damage. Cell growth curve was used to evaluate the growth inhibition of CNE1 by mutant Rad50 and radiation. Results: Expressions of Mre11, Rad50, and Nbs1 in cells of Ad-Rad50-GFP group were less significantly than those in control group when irradiation was completed (0.48 vs 0.62, 0.42 vs 0.5, and 0.53 vs 0.69, respectively, P<0.05) and 24 hours after irradiation (0.41 vs 0.69, 0.46 vs 0.58, and 0.34 vs 0.78, respectively, P<0.05). The mean tail moment (MTM) in Ad-Rad50-GFP plus irradiation group was higher than that in irradiation group when irradiation was completed (16.06 vs 14.8, P<0.05), 24 hours after irradiation (58.23 vs 15.89, P<0.05) and 48 hours after irradiation: (45.12 vs 11.42, P<0.05). Seven days after irradiation, the cells in Ad-Rad50-GFP plus irradiation group was less than those in control group or irradiation group (both P<0.05). Conclusion: Mutant Rad50 enhances killing effects of radiation on nasopharyngeal carcinoma cell line CNE1.

PAKs in Human Cancer Progression: From Inception to Cancer Therapeutic to Future Oncobiology.

Since the initial recognition of a mechanistic role of p21-activated kinase 1 (PAK1) in breast cancer invasion, PAK1 has emerged as one of the widely overexpressed or hyperactivated kinases in human cancer at-large, allowing the PAK family to make in-roads in cancer biology, tumorigenesis, and cancer therapeutics. Much of our current understanding of the PAK family in cancer progression relates to a central role of the PAK family in the integration of cancer-promoting signals from cell membrane receptors as well as function as a key nexus-modifier of complex, cytoplasmic signaling network. Another core aspect of PAK signaling that highlights its importance in cancer progression is through PAK's central role in the cross talk with signaling and interacting proteins, as well as PAK's position as a key player in the phosphorylation of effector substrates to engage downstream components that ultimately leads to the development cancerous phenotypes. Here we provide a comprehensive review of the recent advances in PAK cancer research and its downstream substrates in the context of invasion, nuclear signaling and localization, gene expression, and DNA damage response. We discuss how a deeper understanding of PAK1's pathobiology over the years has widened research interest to the PAK family and human cancer, and positioning the PAK family as a promising cancer therapeutic target either alone or in combination with other therapies. With many landmark findings and leaps in the progress of PAK cancer research since the infancy of this field nearly 20 years ago, we also discuss postulated advances in the coming decade as the PAK family continues to shape the future of oncobiology.

Epigenomic regulation of oncogenesis by chromatin remodeling.

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

Comparative study of clinicopathological significance, BIRC7, and STC2 expression between squamous cell/adenosquamous carcinomas and adenocarcinoma of gallbladder.

Gallbladder cancers (GBCs) are uncommon, but highly aggressive cancers. The majority of GBCs are adenocarcinomas (ACs), but rare subtypes of GBCs such as squamous cell carcinoma (SC) and adenosquamous carcinoma (ASC) are observed as well. The clinicopathological characteristics of SC/ASC have not been well documented. Expressions of BIRC7 and STC2 were observed in some tumors. However, BIRC7 and STC2 expressions and clinical significances in gallbladder cancer have not been reported.In this study, the protein expressions of BIRC7 and STC2 in 46 SCs/ASCs and 80 ACs were measured using immunohistochemistry. We demonstrated that positive BIRC7 and STC2 expressions were significantly associated with large tumor mass (>3cm), high TNM stage and lymph node metastasis in SC/ASC and AC (p<0.05). Positive expression of BIRC7 was significantly associated with invasion of around tissues and organs in both SC/ASC and AC. Additionally, negative BIRC7 and STC2 expressions were significantly associated with surgical curability in AC. Univariate Kaplan-Meier analysis showed that BIRC7 and STC2 expressions, differentiation, tumor size, TNM stages, invasion, lymph node metastasis, and surgical curability were significantly associated with post-operative survival in both SC/ASC and AC patients(p < 0.001). Multivariate Cox regression analysis showed that positive BIRC7 and STC2 expressions are independent poor-prognostic factors in both SC/ASC and AC patients. Our study suggested that positive BIRC7 and STC2 expressions are closely correlated with clinical, pathological, and biological behaviors as well as poor-prognosis of gallbladder cancer.

Potential autocrine regulation of interleukin-33/ST2 signaling of dendritic cells in allergic inflammation.

This study identified a novel phenomenon that dendritic cells (DCs) produced interleukin (IL)-33 via Toll-like receptor (TLR)-mediated innate pathway. Mouse bone marrow-derived DCs were treated with or without microbial pathogens or recombinant murine IL-33. IL-33 mRNA and protein were found to be expressed by DCs and largely induced by several microbial pathogens, highly by lipopolysaccharide (LPS) and flagellin. Using two mouse models of topical challenge by LPS and flagellin and experimental allergic conjunctivitis, IL-33-producing DCs were observed in ocular mucosal surface and the draining cervical lymph nodes in vivo. The increased expression levels of myeloid differentiation primary-response protein 88 (MyD88), nuclear factor (NF)-κB1, NF-κB2, and RelA accompanied by NF-κB p65 nuclear translocation were observed in DCs exposed to flagellin. IL-33 induction by flagellin was significantly blocked by TLR5 antibody or NF-κB inhibitor quinazoline and diminished in DCs from MyD88 knockout mice. IL-33 stimulated the expression of DC maturation markers, CD40 and CD80, and proallergic cytokines and chemokines, OX40L, IL-4, IL-5, IL-13, CCL17 (C-C motif chemokine ligand 17), TNF-α (tumor necrosis factor-α), and IL-1ß. This stimulatory effect of IL-33 in DCs was significantly blocked by ST2 antibody or soluble ST2. Our findings demonstrate that DCs produce IL-33 via TLR/NF-κB signaling pathways, suggesting a molecular mechanism by which local allergic inflammatory response may be amplified by DC-produced IL-33 through potential autocrine regulation.

IL-17RA aptamer-mediated repression of IL-6 inhibits synovium inflammation in a murine model of osteoarthritis.

OBJECTIVE: Generate DNA aptamers to inhibit IL-17RA-mediated synovial inflammation in an experimental mouse model of osteoarthritis (OA). METHODS: A novel cell-SELEX method was applied to obtain DNA aptamers specific for IL-17RA. A single-stranded (ss) DNA library with four(30) probes was synthesised. By incubating this library with NIH3T3 cells, we collected DNA ligands that could bind the cell surface. The collected ligands were incubated with IL-17RA-deficient NIH3T3 cells, and unbound ssDNA was harvested from the supernatant for the next round of selection. After 12 cycles, specific aptamers against IL-17RA were generated. For animal experiments, a meniscectomy was performed on Balb/C mice to generate an animal model of OA. Mice received weekly intra-articular (i.a.) injections of aptamers or control treatments for 6 weeks. Synovial membranes were evaluated by histomorphology and the mRNAs of critical inflammatory cytokines were measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: An aptamer termed RA10-6 was obtained that could efficiently block IL-17 binding to IL-17RA in a dose-dependent manner in vitro. Histological examination and quantitative RT-PCR results showed that OA mice that injected with RA10-6, especially in combination with celecoxib demonstrated inhibition of synovial thickening and reduction in IL-6 levels in the synovial tissue. CONCLUSION: Our results suggest that RA10-6 can inhibit synovial inflammation by blocking IL-17/IL-17RA-mediated IL-6 expression. RA10-6 acted synergistically with celecoxib to inhibit IL-6 expression in synovial tissues. Thus, aptamers targeting IL-17RA might serve as potent adjunctive agents for the early treatment of OA.

TGF-ß1 signaling targets metastasis-associated protein 1, a new effector in epithelial cells.

In spite of a large number of transforming growth factor ß1 (TGF-ß1)-regulated genes, the nature of its targets with roles in transformation continues to be poorly understood. Here, we discovered that TGF-ß1 stimulates transcription of metastasis-associated protein 1 (MTA1), a dual master coregulator, in epithelial cells, and that MTA1 status is a determinant of TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) phenotypes. In addition, we found that MTA1/polymerase II/activator protein-1 (AP-1) co-activator complex interacts with the FosB-gene chromatin and stimulates its transcription, and FosB in turn, utilizes FosB/histone deacetylase 2 complex to repress E-cadherin expression in TGF-ß1-stimulated mammary epithelial cells. These findings suggest that TGF-ß1 regulates the components of EMT via stimulating the expression of MTA1, which in turn, induces FosB to repress E-cadherin expression and thus, revealed an inherent function of MTA1 as a target and effector of TGF-ß1 signaling in epithelial cells.

Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography-mass spectrometry.

An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24h ambient temperature water immersion (ATWE) and 56h ATWE extracts. Two transformation pathways including UMP-->uridine-->uracil and GMP-->guanosine-->guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP-->adenosine-->inosine-->hypoxanthine was proposed in natural C. sinensis, while AMP-->adenosine-->adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis.

Association of ATP1A1 gene polymorphism with heat tolerance traits in dairy cattle.

Heat stress produces oxidative stress and affects the alternation of plasma K(+) and Na(+). Since Na(+),K(+)-ATPase is sensitive to oxidative stress and critical for maintaining the homeostasis of these two ions, we examined the genetic polymorphism of the ATP1A1 gene in 160 Holstein cows using polymerase chain reaction low ionic strength single-strand conformation polymorphism and DNA sequencing methods. G to A at position -14103 in exon 14 and C to T at position -14242 in intron 14 of the bovine ATP1A1 gene were identified, but the former single nucleotide polymorphism was silent with respect to the amino acid sequence of the protein. However, we found significant correlations between ATP1A1 gene polymorphism and the coefficient of heat tolerance (P<0.01) and with respiratory rate (P<0.01). Genotype AC was the most favorable genotype for heat tolerance. This polymorphism site has potential as a genetic marker for heat tolerance traits in dairy cattle breeding.

A simple and evolutional approach proven to recanalise the nasolacrimal duct obstruction.

AIM: To evaluate a new approach of recanalisation of nasolacrimal duct obstruction (RC-NLDO) in the treatment of the nasolacrimal duct obstruction (NLDO) and chronic dacryocystitis. METHODS: 583 patients with 641 eyes suffering from NLDO and chronic dacryocystitis were enrolled in this study. The RC-NLDO was performed in 506 eyes, with 135 eyes undergoing external dacryocystorhinostomy (EX-DCR) as controls. Patient follow-up for 54 months was evaluated by symptoms, dye disappearance test, lacrimal irrigation and digital subtraction dacryocystogram. The RC-NLDO was also performed in 12 rhesus monkeys for histopathological examination. RESULTS: The clinical success rates were 93.1% in 506 cases of RC-NLDO and 91.11% in 135 cases of EX-DCR. The success rates for second surgery were achieved in 85.19% on RC-NLDO and 40.0% on EX-DCR. No major intra- or postoperative complications were observed in the RC-NLDO group. The mean operative duration was 12.5 min for RC-NLDO and 40.3 min for EX-DCR (p<0.001). A pathological study in rhesus monkeys demonstrated that the RC-NLDO wounded epithelium in nasolacrimal duct healed completely within 1 month without granulation tissue formation. CONCLUSION: The findings demonstrate that the RC-NLDO is a simple and effective approach proven to recanalise the obstructed nasolacrimal duct with a comparable success rate to EX-DCR.

PAK signaling in oncogenesis.

The p21-activated kinase (PAK) family of serine/threonine kinases is important in physiological processes including motility, survival, mitosis, transcription and translation. PAKs are evolutionally conserved and widely expressed in a variety of tissues and are often overexpressed in multiple cancer types. Depending on structural and functional similarities, the six members of PAK family are divided into two groups with three members in each group. Group I PAKs are activated by extracellular signals through GTPase-dependent and GTPase-independent mechanisms. In contrast, group II PAKs are constitutively active. Over the years, accumulating data from tissue culture models and human tumors has increased our understanding about the biology of PAK family members. In this review, we have summarized the complex regulation of PAK and its downstream diverse myriads of effectors, which in turn are responsible for the biological effects of PAK family of kinases in cancer cells.

Expression of glial cell-derived neurotrophic factor and its receptor in the stem-cell-containing human limbal epithelium.

AIM: To evaluate the expression pattern of glial cell line-derived neurotrophic factor (GDNF) with its receptors GDNF family receptor alpha-1 (GFR alpha-1) and Ret in the human corneal and limbal tissues, as well as in the primary human limbal epithelial cultures (PHLEC). METHODS: Expression of GDNF and its receptors, and the co-localisation with stem cell associated and differentiation markers were evaluated by immunofluorescent staining, western blot analysis and real-time PCR in the fresh human corneoscleral tissues, as well as in the PHLEC. Single cell colony-forming and wound-healing assays were also evaluated in PHLEC. RESULTS: GDNF and GFR alpha-1 were found to be expressed by a subset of basal cells and co-localised with ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2) and p63, but not with cytokeratin 3 in the human limbal basal epithelium. In PHLEC, they were expressed by a small population of cells in the less differentiated stage. The GDNF and GFR alpha-1-positive subpopulations were enriched for the expression of ABCG2 and p63 (p<0.01). Recombinant human GDNF promoted the proliferation and wound healing of epithelial cells in the PHLEC. In contrast, Ret was abundantly located in the human corneal epithelium except for the basal cells of the limbal epithelium. CONCLUSION: These findings indicate that GDNF and GFR alpha-1 may represent a property for the phenotype of human corneal epithelial precursor cells. GDNF may signal independently of Ret through GFR alpha-1 in the stem cell-containing limbal epithelium.

Serine 28 phosphorylation of NRIF3 confers its co-activator function for estrogen receptor-alpha transactivation.

NRIF3 is an estrogen-inducible nuclear receptor coregulator that stimulates estrogen receptor-alpha (ERalpha) transactivation functions and associates with the endogenous ER and its target gene promoter. p21-activated protein kinase 1 (Pak1) phosphorylates ERalpha at Ser305 and this modification is important in ERalpha transactivation function. Although ERalpha transactivation functions are regulated by co-activator activity of NRIF3, it remains unclear whether Pak1 could impact ER functions via a posttranslational modification of NRIF3. Here, we report that Pak1 phosphorylates NRIF3 at Serine28 and that NRIF3 binds to Pak1 in vitro and in vivo. We found that NRIF3 phosphorylation, co-activator activity and association with ERalpha increased following Pak1 phosphorylation of NRIF3's Ser28 and that activated ERalpha-Ser305 and NRIF3-Ser28 cooperatively support transactivation of ERalpha. NRIF3 expression increased significantly in cells with inducible Pak1 expression. We found that NRIF3 and ERalpha interaction, subcellular localization and ERalpha transactivation activity all increased in cells expressing the Pak1 phosphorylation-mimicking mutant NRIF3-Ser28Glu. Consistently, the NRIF3-Ser28Glu mutant exhibited an enhanced recruitment to the endogenous ER target genes and increased expression following estrogen stimulation. Finally, breast cancer cells with stable overexpression of NRIF3 showed increased proliferation and enhanced anchorage-independent growth. These findings suggest that NRIF3-Ser28 is a physiologic target of Pak1 signaling and contributes to the enhanced NRIF3 co-activator activity, leading to coordinated potentiation of ERalpha transactivation, its target gene expression and estrogen responsiveness of breast cancer cells.

Regulation of MMP-9 production by human corneal epithelial cells.

The matrix metalloproteinases, MMP-2 and MMP-9, are known to be critical extracellular-remodeling enzymes in wound healing and other diseases of the ocular surface. This study investigated the regulation of MMP-2 and MMP-9 in human corneal epithelial cells by growth factors and pro-inflammatory cytokines (IL-1beta and TNF-alpha) they are exposed to, and by doxycycline, a medication used to treat ocular surface disease. Primary human corneal epithelial cell cultures were treated with one of the following cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha) or growth factors (EGF, HGF, KGF, PDGF-BB, TGF-alpha, TGF-beta), with or without their corresponding inhibitors. The conditioned media were collected after 24 hr for gelatin zymography and MMP-9 activity assay. Total RNA was extracted from the cells treated for 6 hr and was subjected to RT-PCR and Northern hybridization. Between the two gelatinases, MMP-2 and MMP-9, detected by zymography, the 92 kDa MMP-9 in the conditioned medium was markedly up-regulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. The MMP-9 protein and activity were dose-dependently stimulated by IL-1beta or TNF-alpha at 0.1, 1.0 and 10 ng ml(-1). This up-regulation was nearly abolished by neutralizing antibodies (IL-1beta and TNF-alpha) and by IL-1 receptor antagonist. Semi-quantitative RT-PCR and Northern hybridization disclosed that the MMP-9 transcript was also markedly up-regulated in a dose-dependent manner by IL-1beta and TNF-alpha. Doxycycline (10 microg ml(-1)) suppressed MMP-9 protein level and activity, but not its mRNA, that was stimulated by IL-1beta and TNF-alpha (1 ng ml(-1)). In contrast, the 72 kDa MMP-2 was not significantly modulated by any of these cytokines. In conclusion, production of MMP-9 is stimulated by the pro-inflammatory cytokines, IL-1beta and TNF-alpha. These factors may play a role in the pathogenesis of MMP-9 mediated corneal matrix degradation. The efficacy of doxycycline in treating ocular surface diseases may be related to its ability to suppress MMP-9 production in the corneal epithelium.

Regulation of collagenase, stromelysin, and gelatinase B in human conjunctival and conjunctivochalasis fibroblasts by interleukin-1beta and tumor necrosis factor-alpha.

PURPOSE: Overexpression and increased activities of matrix metalloproteinases (MMPs) have recently been reported in cultured conjunctival fibroblasts from patients with conjunctivochalasis. The role of inflammatory cytokines in modulating expression of MMPs, their tissue inhibitors (TIMPs), and urokinase plasminogen activator (uPA) as potential contributors to the pathogenesis of conjunctivochalasis was investigated. METHODS: Interleukin-1beta (IL-1beta) or tumor necrosis factor-alpha (TNF-alpha) was added at 10 ng/ml to a serum-free medium. Expression of transcripts and proteins of MMPs, TIMPs, and uPA by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. Gelatin and casein zymographies were performed in serum-free conditioned media with and without the respective enzyme inhibitors. RESULTS: Without challenging the cells, conjunctivochalasis fibroblasts showed mRNA and protein overexpression of MMP-1 and MMP-3 compared with normal conjunctival fibroblasts, which showed minor or no expression of these enzymes. IL-1beta markedly and TNF-alpha to lesser extent increased mRNA and protein expression of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts from 2 subjects when compared with normal conjunctival fibroblasts from 2 subjects and with their nonstimulated counterparts. In conjunctivochalasis fibroblasts and normal conjunctival fibroblasts, TNF-alpha, but not IL-1beta, induced a gelatinolytic activity of MMP-9, which was further confirmed by Western blot analysis and ELISA. Expression of MMP-2, TIMP-1, and TIMP-2 mRNA and protein was not influenced by IL-1beta or TNF-alpha, and no difference was found in the gelatinolytic activity of MMP-2 between both cell types. CONCLUSIONS: Inflammatory cytokines such as IL-1beta and TNF-alpha, which can potentially be derived from the ocular surface and tears, may be responsible for increased expression of MMPs in cultured conjunctivochalasis fibroblasts. Ocular inflammation might be one important denominator in the pathogenesis of conjunctivochalasis.