Alcohol inhibits osteopontin-dependent transforming growth factor-ß1 expression in human mesenchymal stem cells.

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor ß1 (TGF-ß1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-ß1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-ß1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-ß1 protein expression, decreases MZF1-dependent TGF-ß1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-ß1 expression. Last, we showed that EtOH exposure reduces the TGF-ß1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-ß1 signaling pathway in MSC.

Tumor: Stroma Interaction and Cancer.

The understanding of how normal cells transform into tumor cells and progress to invasive cancer and metastases continues to evolve. The tumor mass is comprised of a heterogeneous population of cells that include recruited host immune cells, stromal cells, matrix components, and endothelial cells. This tumor microenvironment plays a fundamental role in the acquisition of hallmark traits, and has been the intense focus of current research. A key regulatory mechanism triggered by these tumor-stroma interactions includes processes that resemble epithelial-mesenchymal transition, a physiologic program that allows a polarized epithelial cell to undergo biochemical and cellular changes and adopt mesenchymal cell characteristics. These cellular adaptations facilitate enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components. Indeed, it has been postulated that cancer cells undergo epithelial-mesenchymal transition to invade and metastasize.In the following discussion, the physiology of chronic inflammation, wound healing, fibrosis, and tumor invasion will be explored. The key regulatory cytokines, transforming growth factor ß and osteopontin, and their roles in cancer metastasis will be highlighted.

Long-term outcomes after simultaneous pancreas-kidney transplant.

PURPOSE OF REVIEW: Simultaneous pancreas-kidney (SPK) transplantation represents the only proven long-term therapeutic approach for type 1 diabetic, dialysis-dependent patients. This procedure potentially liberates these patients from dialysis and the need for exogenous insulin replacement. For the first time, data on the long-term natural history of patients receiving SPK have recently been analyzed. In this review, we discuss the outcomes and complications for patients receiving SPK in the context of the current literature. RECENT FINDINGS: In our analysis of 1000 SPKs performed at our center, we demonstrated that SPK increases patient survival compared with live-donor kidney alone or deceased donor kidney alone transplantation. The 5-year, 10-year, and 20-year patient survival for SPK recipients was 89, 80, and 58%, respectively. Enteric drainage improves quality of life, but not allograft survival, when compared with bladder drainage. After transplantation, approximately 50% of bladder-drained transplants undergo enteric conversion and late conversion after transplantation is associated with a higher complication rate. Surgical complications are higher in enteric-drained compared with bladder-drained pancreas transplants. SUMMARY: Selecting the appropriate therapy for a type 1 diabetic recipient with renal failure continues to be a critical decision for programs offering pancreas transplantation. The principles and guidelines at our center are driven by the potential benefit of the SPK transplant needing to outweigh the increased morbidity of the surgical procedure and the use of lifelong immunosuppression. Results from long-term studies demonstrating improved patient survival suggest that the treatment of choice for an appropriate type 1 diabetic recipient is an SPK transplant.

Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4alpha transcriptional activity.

In IL-1beta (interleukin 1beta)-stimulated rat hepatocytes exposed to superoxide, we have previously identified an IRX (inflammatory redox)-sensitive DR1 [direct repeat of RG(G/T)TCA with one base spacing] cis-acting activator element (nt -1327 to -1315) in the iNOS (inducible nitric oxide synthase) promoter: AGGTCAGGGGACA. The corresponding transcription factor was identified to be HNF4alpha (hepatocyte nuclear factor-4alpha). HNF4alpha DNA binding activity and transactivation potential are tightly regulated by its state of phosphorylation. However, the functional consequences of IRX-mediated post-translational phosphorylation of HNF4alpha have not been well characterized. In the setting of IL-1beta+H2O2, HNF4alpha functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4alpha to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4alpha, serial mutations were performed to render the target residues phosphorylation-resistant. Electrophoretic mobility-shift assays and transient transfection studies utilizing the iNOS promoter showed that the S158A mutation ablates IRX-mediated HNF4alpha DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4alpha vector supports a critical role for Ser158 phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser158 phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4alpha in the presence of IL-1beta+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.

Osteopontin inhibits macrophage nitric oxide synthesis to enhance tumor proliferation.

BACKGROUND: Interactions between tumor cells and their host environment can play a major role in regulating survival programs required for tumor progression. Osteopontin (OPN) is a glycophosphoprotein overexpressed by tumors, and is a key molecule for tumor progression and metastasis. OPN also inhibits expression of autocrine and paracrine inducible nitric oxide synthase (iNOS). Given the cytotoxic effects of macrophage NO expression, we hypothesized that tumor-derived OPN inhibits expression of local macrophage iNOS to potentiate tumor survival. METHODS: We used a coculture system of murine CT26 colorectal cancer cells with RAW264.7 murine macrophage cells. CT26 expresses OPN at high levels. RNA interference was utilized to produce long-term specific silencing of OPN in CT26. RESULTS: Inhibition of constitutive OPN synthesis in CT26 upregulates local NO production with inhibition of CT26 proliferation and promotion of CT26 apoptosis. Macrophage iNOS expression is accompanied by increased binding activity of nuclear factor-kappaB DNA. When the CT26 culture media were examined for a panel of proinflammatory cytokines, elevated concentrations of granulocyte colony-stimulating factor (G-CSF) were found. Subsequently, in CT26 cells treated with antisense-G-CSF, NO levels in CT26-RAW cocultures were significantly decreased. CONCLUSION: In our system of CT26-RAW264.7 coculture, we conclude that inhibition of OPN synthesis in CT26 results in G-CSF-mediated induction of macrophage iNOS expression with resultant inhibition of CT26 proliferation via increased apoptosis. Our results suggest that tumor-derived OPN may enhance tumor survival by down regulating expression of NO in the local microenvironment. This is one mechanism by which OPN may potentiate cancer survival and progression.

Redox-mediated upregulation of hepatocyte iNOS transcription requires coactivator PC4.

BACKGROUND: Redox-mediated upregulation of transcription of hepatocyte inducible nitric oxide synthase (iNOS) requires hepatocyte nuclear factor IV-alpha (HNF-4alpha). In this setting, PC4 is often isolated with HNF-4alpha in DNA-protein pull-down studies. Transcriptional coactivator PC4 facilitates activator-dependent transcription via interactions with basal transcriptional machinery that are independent of PC4-DNA binding. We hypothesized that PC4 is a necessary component of HNF-4alpha-regulated redox-sensitive hepatocyte iNOS transcription. METHODS: Murine CCL9.1 hepatocytes were stimulated with interleukin-1beta (IL-1beta; 1000 U/mL) in the presence and absence of peroxide (H(2)O(2); 50 nmol/L). Antisense and sense oligonucleotides to HNF-4alpha and PC4 were added selectively. Coimmunoprecipitation (Co-IP) studies determined the association between HNF-4alpha and PC4. Transient transfection was performed with the use of a luciferase reporter construct containing the murine iNOS promoter (1.8 kb). Chromatin immunoprecipitation assays determined in vivo binding of PC4 and HNF-4alpha to the iNOS promoter region. RESULTS: Ablation of either HNF-4alpha or PC4 blunted the peroxide-mediated increase in the activation of the iNOS promoter. In IL-1beta+H(2)O(2) only, co-IP studies demonstrated the presence of an HNF-4alpha-PC4 protein complex, and chromatin immunoprecipitation assays demonstrated that this complex binds to the genomic iNOS promoter. CONCLUSIONS: Redox-mediated upregulation of hepatocyte iNOS transcription requires an HNF-4alpha-PC4 transcriptional complex.

Doing well by doing good: linking access with quality.

BACKGROUND: We hypothesize that medical centers that prioritize altruism can also deliver superior quality care. METHODS: Data were obtained from California's Office of Statewide Health Planning and Development, Medicare Hospital Compare, and the Joint Commission US Census Bureau's American Community Survey. Outcomes were measured using summary statistics, regression analysis, and quality indices. Total discounted revenue/total revenue (TDR/TR) served as a proxy for altruistic care. RESULTS: In nonprofit hospitals, TDR/TR positively correlated with 5 quality indices including pneumonia (P < .001), heart failure (P = .05), and overall surgical process of care (P = .009). Hospital size predicted higher quality surgical process (P = .06, 201 to 300 beds; P = .01, >301 beds), hospital teaching status demonstrated positive correlation (ß = .048, P = .69), and poverty was negatively correlated (ß = -.00072, P = .89). Positive TDR/TR did not adversely affect mortality or readmission rates (P = .52). CONCLUSIONS: TDR/TR predicts quality in nonprofit hospitals without increasing mortality and readmission. Altruistic motivation may be associated with the delivery of higher quality surgical care.

Engagement, workplace satisfaction, and retention of surgical specialists in academic medicine in the United States.

BACKGROUND: Academic medical centers strive for clinical excellence with operational efficiency and financial solvency, which requires institutions to retain productive and skillful surgical specialists. Faculty workplace perceptions, overall satisfaction, and intent to leave are relationships that have not been examined previously among US surgeons in academic medicine. We hypothesize that critical factors related to workplace satisfaction and engagement could be identified as important for enhancing institutional retention of academic surgeons. STUDY DESIGN: The 2011-2012 Association of American Medical Colleges Faculty Forward Engagement Survey evaluated demographic variables, physician workplace satisfaction, and overall engagement among faculty subgroups, including comparison of surgical and nonsurgical clinicians. Multiple regression analysis (ß = standard regression coefficient) was performed to identify critical factors most closely related to surgeon satisfaction and intent to leave their institutions. RESULTS: A total of 1,356 of 1,949 (70%) surgeons from 14 medical schools responded across different faculty subgroups, and comparisons were made with 1,105 nonsurgical clinicians. Multiple regression indicated that the strongest predictors of surgeons' overall satisfaction with their department included department governance (ß = 0.36; p < 0.001), collegiality and collaboration (ß = 0.23; p < 0.001), and relationship with supervisor (ß = 0.17; p < 0.001). Although compensation and benefits were important (ß = 0.08; p < 0.001), these did not rank as the most important factors. Promotion equality (odds ratio = 0.62; p < 0.05), collegiality and collaboration (odds ratio = 0 .51; p < 0.05), and nature of their work (odds ratio = 0.52; p < 0.05) were most closely related to intent to leave the medical school within 1 to 2 years. CONCLUSIONS: In the largest survey focusing on workplace factors affecting surgical faculty satisfaction and intent to leave, we conclude that institutional understanding of, and improvement in, specific work environment factors can enhance recruitment and retention of academic surgeons.

Comparing 20 years of national general surgery malpractice claims data: obesity versus morbid obesity.

BACKGROUND: We hypothesized that the increasing body mass index of the population has affected general surgery malpractice claims. METHODS: We queried the Physician Insurers Association of America database from 1990 to 1999 (ie, period 1) and 2000 to 2009 (ie, period 2) for claims associated with obesity and morbid obesity. We analyzed the error involved, injury severity, procedure, and outcome. RESULTS: Five hundred seventy-five claims were identified. The percentage of paid claims did not differ by body mass index. Improper performance was the most common alleged error, gastric bypass was the most common procedure, and death was the most common injury. For obesity claims, the case was more likely to be settled in period 1 and withdrawn/dismissed in period 2 (P < .001). The number of morbid obesity claims rose from 9 in period 1 to 249 in period 2. CONCLUSIONS: The significant rise in morbid obesity claims between periods is likely caused by the substantial increase in the number of bariatric procedures performed.

Osteopontin up-regulates critical epithelial-mesenchymal transition transcription factors to induce an aggressive breast cancer phenotype.

BACKGROUND: Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPN's role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPN's role in Twist activation is examined through immunoprecipitation and Western blot. STUDY DESIGN: MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation. RESULTS: Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN+APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression. CONCLUSIONS: This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis.

Obesity and trends in malpractice claims for physicians and surgeons.

BACKGROUND: The increasing prevalence of obesity has altered the practice of medicine and surgery, with the emergence of new operations and medications. We hypothesized that the landscape of medical malpractice claims has also changed. METHODS: We queried the Physician Insurers Association of American database for 1990 through 1999 and 2000 through 2009 for cases corresponding to International Classification of Diseases, 9th edition, codes for obesity. We extracted adjudicatory outcome, closed and paid claims data, indemnity payments, primary alleged error codes, National Association of Insurance Commissioners severity of injury class, procedural codes, and medical specialty data. RESULTS: A total of 411 obesity claims were filed from 1990 to 1999 and 1,591 obesity claims were filed from 2000 to 2009. General surgery was the specialty with the greatest number of obesity claims from 1990 to 1999 and was second to family practice for 2000 to 2009. Although the percentage of paid general surgery obesity claims has decreased significantly from 69% in 1990-1999 to 36% in 2000-2009, the mean indemnity payments have increased substantially ($94,000 to $368,000). CONCLUSION: Recently, the percentage of paid general surgery obesity claims has significantly decreased; however, individual and total indemnity payments have increased. Obesity continues to impact general surgery malpractice substantially. Efforts to manage this component of physician and hospital practices must continue.

An MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines.

BACKGROUND: Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer. METHODS: Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment. RESULTS: MEK1/2 inhibition caused ≈ twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001). CONCLUSION: We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.

Intersecting pathways in inflammation and cancer: Hepatocellular carcinoma as a paradigm.

Viral infection and chemical carcinogens trigger somatic changes resulting in activation of oncogenes during tumor initiation in the development of cancer. However, a critical interaction resides in the synergism between these somatic changes and an inflamed tumor microenvironment where myeloid and hematopoietic cells are subverted to enhance tumor progression. The causative molecular mechanisms leading to the development of hepatocellular cancer remain incompletely understood but appear to result from multiple factors related to direct hepatocyte injury and the ensuing inflammatory changes mediated by the host response to tissue injury, DNA damage, repair of cellular damage, and chronic, repetitive injury. In this review, the molecular and cellular changes that regulate inflammation and tissue repair will be compared to the activated local tumor microenvironment. Cell-cell signaling within this microenvironment that enhances tumor progression and inhibits anti-tumor immunity will be discussed.

Epithelial-mesenchymal transition, TGF-ß, and osteopontin in wound healing and tissue remodeling after injury.

Epithelial-mesenchymal transition (EMT) is a process essential to wound healing and tissue remodeling after a thermal burn or other injury. EMT is characterized by phenotypic changes in epithelial cells that render them apolar, with decreased cell-cell adhesions, increased motility, and changes in cytoskeletal architecture similar to mesenchymal stem cells. With regard to healing a thermal burn wound, many facets of wound healing necessitate cells to undergo these phenotypic changes; two will be described in the following review. The first is the differentiation of epithelial cells into myofibroblasts that rebuild the extracellular matrix and facilitate wound contraction. The second is reepithelialization by keratinocytes. The primary cytokine signal identified in the literature that triggers EMT is transforming growth factor (TGF)-ß. In addition to its vital role in the induction of EMT, TGF-ß has many other roles in the wound healing process. The following review will provide evidence that EMT is a central event in wound healing. It will also show the importance of a regulated amount of TGF-ß for proper wound healing. Finally, osteopontin will be briefly discussed with its relation to wound healing and its connections to EMT and TGF-ß.

Impact of business infrastructure on financial metrics in departments of surgery.

BACKGROUND: In the current environment, pressure is ever increasing to maximize financial performance in surgery departments. Factors such as physician extenders, billing and collection, payor mix, contracting, incentives from the Centers for Medicare and Medicaid Services, and administrative incentives may greatly influence financial performance. However, despite a plethora of information from the University HealthSystem Consortium and the Association of American Medical Colleges, best-practice information for business infrastructure is lacking. To obtain a sampling of current practices, we conducted a survey of departments of surgery. METHODS: An anonymous 30-question survey addressing demographics, productivity, revenue and expense profile, payor mix, physician extender and staff personnel, billing and collections methodology, and financial performance was distributed among members of the Society of Surgical Chairs via SurveyMonkey. This was approved by the Loyola Institutional Research Board. Multivariate linear regression analyses and t tests/rank-sum tests were performed, as appropriate. Data are presented as mean ± SEM. RESULTS: A total of 25 (19%) departments responded; 14 were integrated with the hospital/health system, and 11 were integrated with the medical school. In 60% (n = 15), the main hospital had 500 to 1,000 beds; 48% (n = 12) had >4 hospitals in their system. For FY10, MD clinical full-time equivalents (FTEs) were 49 ± 10; total work relative value units (wRVUs) were 320 ± 8 k; and total billed cases were 43 ± 16 k. A total of 23 of 25 used physician-extenders with an average of 18 ± 5 per department and in 22 of 23, the physician extenders billed. On average, there were 18 ± 6 clinical-support staff, 25 ± 11 front-office staff, and 13 ± 3 back-office support staff FTEs. Among these FTEs, there were 16 ± 5 devoted to business operations (billing, coding, denial/claims management, financial oversight). Collections/wRVUs were $60 ± 3 (range, 39-80). Regression modeling demonstrated that total wRVUs were determined by the number of MD FTEs (P = .01), number of physician extenders (P = .01), number of front-office staff (P = .01), number of back-office staff (P = .02), and number of total business staff (P = .01). Collections/wRVUs were predicted by number of hospitals (P = .04), number of MD FTEs (P = .03), number of physician extenders (P = .01), and number of cases/total business staff (P = .02). Interestingly, wRVUs/MD was predicted by number of MD FTEs (P = .01) but were not greatly impacted by numbers of clinical or business support staff. In 4 of 25, the billing and coding staff were incentivized and had a Collections/wRVU = 64 ± 5 whereas nonincentivized staff had collections/wRVU = 59 ± 3. (P = NS) Also, %Accounts receivable >90 days (15% vs 25%) were not substantially different. Only 48% (12/25) have departments have recouped Centers for Medicare and Medicaid dollars for Physician Quality Reporting Initiative, Meaningful Use, Patient-Centered Medical Homes, or other Accountable Care-like programs. One-half (13) of the departments had both an inpatient and outpatient electronic medical record. Finally, on a scale of 1-10 (10 = highest), the average level of satisfaction with billing and collections processes was 6. CONCLUSION: Our results indicate that the physician extender, clinical support staff, and business staff environment can impact surgeon productivity, and there is opportunity for improvement. Determining best practices for ratios of support staff/MD and optimizing the role of electronic medical record in workflow and billing/collections are critical in the current environment. Our pilot study requires extension across more institutions for validation.

Osteopontin: regulation in tumor metastasis.

Osteopontin is a secreted phosphoprotein that has been implicated as an important mediator of tumor metastasis and has been investigated for use as a biomarker for advanced disease and as a potential therapeutic target in the regulation of cancer metastasis. The OPN DNA sequence is highly conserved and the protein contains several important functional domains including alpha(v)beta integrin and CD44 binding sites. High levels of OPN expression correlate with tumor invasion, progression or metastasis in multiple cancer. Studies demonstrate that osteopontin mediates the molecular mechanisms which determine metastatic spread, such as prevention of apoptosis, extracellular matrix proteolysis and remodeling, cell migration, evasion of host-immune cells and neovascularization. Transcriptional regulation of OPN is complex and involves multiple pathways, including AP-1, Myc, v-Src, Runx/CBF, TGF-B/BMPs/Smad/Hox, and Wnt/ss-catenin/APC/GSK-3ss/Tcf-4. The current state of knowledge of OPN biology suggests that it is an attractive target for therapeutic modulation of metastatic disease.

Osteopontin mediates Stat1 degradation to inhibit iNOS transcription in a cecal ligation and puncture model of sepsis.

BACKGROUND: Osteopontin (OPN) represses inducible nitric oxide synthase (iNOS) expression by increasing ubiquitin (Ub)-proteasome degradation of Stat1, a critical transcription factor for iNOS expression. We investigated the in vivo relevance of our findings in a cecal ligation and puncture model. METHODS AND RESULTS: A total of 129 wild-type (WT; n = 24) and OPN null (n = 24) mice were used. Bone marrow macrophages and whole liver tissue were isolated. iNOS and phosphorylated Stat-1 (P-Stat1) protein were significantly greater in OPN null than WT. Cecal ligation and puncture increased Ub-P-Stat1; Ub-P-Stat1 was significantly less in OPN null than WT. In chromatin immunoprecipitation assays, P-Stat1 binding to the iNOS promoter was increased in OPN null. Ex vivo studies with bone marrow macrophages were performed with MG132 (10 microM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 microM). Ub-P-Stat1 was decreased in OPN null bone marrow macrophages treated with LPS; iNOS was increased. Exogenous OPN or MG132 restored Ub-P-Stat1 and iNOS to levels seen in WT. Our results indicate that absence of OPN does the following: (1) increases iNOS and P-Stat1 protein, (2) decreases ubiquitination and degradation of P-Stat1, and (3) increases iNOS transcription. CONCLUSIONS: We conclude that OPN downregulates iNOS expression by accelerating ubiquitination and degradation of Stat1.

Ets-1 and runx2 regulate transcription of a metastatic gene, osteopontin, in murine colorectal cancer cells.

Osteopontin (OPN) is a sialic acid-rich phosphoprotein secreted by a wide variety of cancers. We have shown previously that OPN is necessary for mediating hepatic metastasis in CT26 colorectal cancer cells. Although a variety of stimuli can induce OPN, the molecular mechanisms that regulate OPN gene transcription in colorectal cancer are unknown. We hypothesized that cis- and trans-regulatory elements determine OPN transcription in CT26 cells. OPN transcription was analyzed in CT26 cancer cells and compared with YAMC (young adult mouse colon) epithelial cells. Clonal deletion analysis of OPN promoter-luciferase constructs identified cis-regulatory regions. A specific promoter region, nucleotide (nt) -107 to -174, demonstrated a >8.0-fold increase in luciferase activity in CT26 compared with YAMC. Gel-shift assays sublocalized two cis-regulatory regions, nt -101 to -123 and nt -121 to -145, which specifically bind CT26 nuclear proteins. Competition with unlabeled mutant oligonucleotides revealed that the regions nt -115 to -118 and nt -129 to -134 were essential for protein binding. Subsequent supershift and chromatin immunoprecipitation assays confirmed the corresponding nuclear proteins to be Ets-1 and Runx2. Functional relevance was demonstrated through mutations in the Ets-1 and Runx2 consensus binding sites resulting in >60% decrease in OPN transcription. Ets-1 and Runx2 protein expression in CT26 was ablated using antisense oligonucleotides and resulted in a >7-fold decrease in OPN protein expression. Ets-1 and Runx2 are critical transcriptional regulators of OPN expression in CT26 colorectal cancer cells. Suppression of these transcription factors results in significant down-regulation of the OPN metastasis protein.

Integrin-linked kinase regulates osteopontin-dependent MMP-2 and uPA expression to convey metastatic function in murine mammary epithelial cancer cells.

Metastasis-supporting physiological alterations are regulated by cell signaling molecules, which target signal transduction pathways and gene expression. Osteopontin (OPN) overexpression may represent a key molecular event in cancer metastasis. In this study, using metastatic 4T1 and non-metastatic 4T07 murine mammary cancer cell lines, we demonstrate that 4T1 cells exhibit significantly increased OPN, integrin-linked kinase (ILK), matrix metalloproteinase-2 (MMP-2) and urokinase-type plasminogen activator (uPA) expression in contrast to 4T07 cells. Blockade of OPN binding to 4T1 cell-surface integrins by the competitive ligand inhibitor, RGD, or a blocking antibody to alphavbeta3 integrin decreases OPN, ILK, MMP-2 and uPA expression. Conversely, exposure of 4T07 cells to exogenous OPN increases ILK, MMP-2 and uPA levels. Further experiments demonstrate that OPN-alphavbeta3 integrin binding in 4T1 with subsequent activation of ILK results in binding of AP-1 to MMP-2 and uPA promoter and increased in vitro promoter activation, as measured by transient transfection assays using MMP-2 and uPA promoter-reporter constructs. AP-1 activity is ablated by co-transfection of DN-ILK or exposure to RGD. Finally, functional correlative assays demonstrate that inhibition of ILK activity or RGD-mediated blockade of alphavbeta3 integrin binding significantly inhibits in vitro invasion, migration and invasion properties of 4T1 cells. In addition, uPA and MMP-2 have overlapping contributions to 4T1 migration and invasion characteristics. However, OPN and ILK activities contribute to 4T1 adhesion activities via mechanisms that are independent of uPA and MMP-2. Our results indicate that binding of an RGD-bearing ligand, such as OPN, to integrin receptors in metastatic 4T1 cells transcriptionally mediates MMP-2, uPA and OPN expression through ILK-dependent AP-1 activity and significantly increases in vitro functional correlates of metastasis. In 4T1 murine mammary cancer cells, we conclude that OPN mediates metastatic behavior, in part, through upregulation of MMP-2 and uPA protein expression.