Correction: da Costa et al. Establishment of a Temperature-Sensitive Model of Oncogene-Induced Senescence in Angiosarcoma Cells. Cancers 2020, 12, 395.

The authors wish to make the following corrections to this paper [...].

Establishment of a Temperature-Sensitive Model of Oncogene-Induced Senescence in Angiosarcoma Cells.

: Lesions with driver mutations, including atypical nevi and seborrheic keratoses, are very common in dermatology, and are prone to senescence. The molecular events that prevent senescent lesions from becoming malignant are not well understood. We have developed a model of vascular proliferation using a temperaturesensitive, large T antigen and oncogenic HRas. By elevating the temperature to 39 °C, we can turn off large T antigen and study the molecular events in cells with the Ras driver mutation. To assess the signaling events associated with the switch from a proliferative to a nonproliferative state in the constant presence of a driver oncogene, SVR cells were cultivated for 24 and 48 hours and compared with SVR cells at 37 °C. Cells were evaluated by Western Blot (WB) gene chip microarray (GC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Upon evaluation, a novel phenotype was observed in endothelial cells after switching off the large T antigen. This phenotype was characterized by Notch activation, downregulation of p38 phosphorylation, downregulation of the master immune switch IRF7, and downregulation of hnRNP A0 . Switching off proliferative signaling may result in immune privilege and Notch activation, which may account, in part, for the survival of common skin lesions.

Tris DBA palladium is an orally available inhibitor of GNAQ mutant uveal melanoma in vivo.

Uveal melanoma is a rare but often lethal malignancy and is the leading cause of death due to an ophthalmic condition. Uveal melanoma is often diagnosed at a late stage and has a strong propensity to hepatic metastasis. Recently, the most common driver mutations in uveal melanoma have been identified, predominantly in the G-proteins GNAQ. This pattern differs from that of cutaneous melanoma in which Braf and Nras predominate. There are no current clinically used agents that target GNAQ mutations, unlike the use of Braf inhibitors in cutaneous melanoma. We tested the novel agent Tris DBA palladium and found that it was markedly more effective against GNAQ mutant melanomas than wild type uveal melanomas. Given that ARF6 has recently been discovered as a node in GNAQ mutations, we evaluated the efficacy of Tris DBA palladium on ARF6 signaling and found that it was effective in inhibiting ARF6 activation. Finally, Tris DBA palladium was orally effective against GNAQ mutant melanoma in vivo. Tris DBA Palladium deserves further evaluation as a systemic agent for uveal melanoma.

Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis.

Psoriasis is a chronic inflammatory skin disease affecting 2.5-6 million patients in the United States. The cause of psoriasis remains unknown. Previous human and animal studies suggest that patients with a susceptible genetic background and some stimulus, such as barrier disruption, leads to a coordinated signaling events involving cytokines between keratinocytes, endothelial cells, T cells, macrophages and dendritic cells. Ceramides are endogenous skin lipids essential for maintaining skin barrier function and loss of ceramides may underlie inflammatory and premalignant skin. Ceramides act as a double-edged sword, promoting normal skin homeostasis in the native state, but can be metabolized to sphingosine-1-phosphate (S1P), linked to inflammation and tumorigenesis. To overcome this difficulty, we synthesized solenopsin analogs which biochemically act as ceramides, but cannot be metabolized to S1P. We assess their in vivo bioactivity in a well-established mouse model of psoriasis, the KC-Tie2 mouse. Topical solenopsin derivatives normalized cutaneous hyperplasia in this model, decreased T cell infiltration, interleukin (IL)-22 transcription, and reversed the upregulation of calprotectin and Toll-like receptor (TLR) 4 in inflamed skin. Finally, they stimulated interleukin (IL)-12 production in skin dendritic cells. Thus suggesting barrier restoration has both a biochemical and physical component, and both are necessary for optimal barrier restoration.

Targeting the duality of cancer.

Cancer is the second leading cause of death in the United States, and is an increasing cause of death in the developing world. While there is great heterogeneity in the anatomic site and mutations involved in human cancer, there are common features, including immortal growth, angiogenesis, apoptosis evasion, and other features, that are common to most if not all cancers. However, new features of human cancers have been found as a result of clinical use of novel "targeted therapies," angiogenesis inhibitors, and immunotherapies, including checkpoint inhibitors. These findings indicate that cancer is a moving target, which can change signaling and metabolic features based upon the therapies offered. It is well-known that there is significant heterogeneity within a tumor and it is possible that treatment might reduce the heterogeneity as a tumor adapts to therapy and, thus, a tumor might be synchronized, even if there is no major clinical response. Understanding this concept is important, as concurrent and sequential therapies might lead to improved tumor responses and cures. We posit that the repertoire of tumor responses is both predictable and limited, thus giving hope that eventually we can be more effective against solid tumors. Currently, among solid tumors, we observe a response of 1/3 of tumors to immunotherapy, perhaps less to angiogenesis inhibition, a varied response to targeted therapies, with relapse and resistance being the rule, and a large fraction being insensitive to all of these therapies, thus requiring the older therapies of chemotherapy, surgery, and radiation. Tumor phenotypes can be seen as a continuum between binary extremes, which will be discussed further. The biology of cancer is undoubtedly more complex than duality, but thinking of cancer as a duality may help scientists and oncologists discover optimal treatments that can be given either simultaneously or sequentially.

Double Jeopardy: The Rubber Ball Bounces Twice.

Soblet et al. describe cis mutations in TEK/Tie-2 in blue rubber bleb nevus and sporadic vascular malformations. This suggests that the remaining normal allele is required for the phenotype. Second, it suggests therapeutic approaches to treatment signal transduction inhibition.

Impact of Phosphorus-Based Food Additives on Bone and Mineral Metabolism.

CONTEXT: Phosphorus-based food additives can substantially increase total phosphorus intake per day, but the effect of these additives on endocrine factors regulating bone and mineral metabolism is unclear. OBJECTIVE: This study aimed to examine the effect of phosphorus additives on markers of bone and mineral metabolism. Design and Setting, and Participants: This was a feeding study of 10 healthy individuals fed a diet providing ∼1000 mg of phosphorus/d using foods known to be free of phosphorus additives for 1 week (low-additive diet), immediately followed by a diet containing identical food items; however, the foods contained phosphorus additives (additive-enhanced diet). Parallel studies were conducted in animals fed low- (0.2%) and high- (1.8%) phosphorus diets for 5 or 15 weeks. MAIN OUTCOME MEASURES: The changes in markers of mineral metabolism after each diet period were measured. RESULTS: Participants were 32 ± 8 years old, 30% male, and 70% black. The measured phosphorus content of the additive-enhanced diet was 606 ± 125 mg higher than the low-additive diet (P < .001). After 1 week of the low-additive diet, consuming the additive-enhanced diet for 1 week significantly increased circulating fibroblast growth factor 23 (FGF23), osteopontin, and osteocalcin concentrations by 23, 10, and 11%, respectively, and decreased mean sclerostin concentrations (P < .05 for all). Similarly, high-phosphorus diets in mice significantly increased blood FGF23, osteopontin and osteocalcin, lowered sclerostin, and decreased bone mineral density (P < .05 for all). CONCLUSIONS: The enhanced phosphorus content of processed foods can disturb bone and mineral metabolism in humans. The results of the animal studies suggest that this may compromise bone health.

A new regulator of osteoclastogenesis: estrogen response element-binding protein in bone.

The heterogeneous nuclear ribonucleoprotein (hnRNP)-like estrogen response element-binding protein (ERE-BP) competes with estrogen receptor α (ERα) for occupancy of estrogen response elements (EREs). Here we report that ERE-BP potently stimulates osteoclastogenesis. ERE-BP mRNA and protein were found to be expressed ubiquitously in bone. Overexpression of ERE-BP in cultured osteoblasts stimulated expression of the receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin (OPG). The effect of ERE-BP on RANKL was shown to be transcriptional in transient transfection assay and competed with via the ER. Constitutive expression of ERE-BP increased the sensitivity of cells toward 1,25-dihydroxyvitamin D(3) stimulation of RANKL expression. In contrast, knockdown of ERE-BP in stromal ST-2 cells decreased basal RANKL promoter activity. Cocultures of ERE-BP lentivirus-transduced ST-2 cells with spleen monocytes induced formation of multinucleated osteoclasts (OCs) characterized by tartrate-resistant acid phosphatase, calcitonin receptors, and functional calcium resorption from bone slices. Although ERα competed with ERE-BP for an ERE in a dose-dependent manner, ERE-BP was an independent and potent regulator of RANKL and osteoclastogenesis. In preosteoclastic RAW cells, overexpression of ERE-BP increased RANK, upregulated NF-κB signaling, and enhanced differentiation toward a mature OC phenotype independent of RANKL. These results identify ERE-BP as a potent modulator of osteoclastogenesis. We hypothesize that ERE-BP may play a critical role in the regulation of bone homeostasis as a modulator of estrogen sensitivity as well as by direct action on the transcription of critical osteoclastogenic genes.

Identification of the homeobox protein Prx1 (MHox, Prrx-1) as a regulator of osterix expression and mediator of tumor necrosis factor α action in osteoblast differentiation.

Tumor necrosis factor α (TNF-α) promotes bone loss and inhibits bone formation. Osterix (Osx, SP7) is a transcription factor required for osteoblast (OB) differentiation because deletion results in a cartilaginous skeleton. We previously described a TNF suppressor element in the Osx promoter that was used to isolate nuclear proteins mediating TNF inhibition of OB differentiation. Nuclear extracts from TNF-treated pre-OBs were incubated with the TNF suppressor element for protein pull-down, and tryptic fragments were analyzed by mass spectrometry. Chromatin immunoprecipitation (ChIP) assay confirmed eight bound transcription factors. One protein, the paired related homeobox protein (Prx1), had been shown previously to have a critical role in limb bud formation and skeletal patterning. PCR revealed Prx1 expression in primary stromal cells (MSCs), C3H10T1/2 cells, and MC3T3 preosteoblasts. TNF stimulated a 14-fold increase in mRNA for Prx1, rapid cell accumulation in MC3T3 cells, and expression in periosteal and trabecular lining cells in vivo. Transient expression of Prx inhibited transcription of Osx and RUNX2. Expression of the Prx1b isoform or Prx2 decreased Osx and RUNX2 mRNA and OB differentiation in preosteoblasts. Silencing of Prx1 with siRNA abrogated TNF suppression of Osx mRNA and increased basal Osx expression. Electrophoretic mobility shift revealed Prx1b as the preferred isoform binding the Osx promoter. These results identify the homeobox protein Prx1 as an obligate mediator of TNF inhibition of Osx and differentiation of OB progenitors. Activation of Prx1 by TNF may contribute to reduced bone formation in inflammatory arthritis, menopause, and aging.

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

The muscle transcription factor MyoD promotes osteoblast differentiation by stimulation of the Osterix promoter.

Transcription factors regulate tissue-specific differentiation of pluripotent mesenchyme to osteoblast (OB), myoblast (MB), and other lineages. Osterix (Osx) is an essential transcription factor for bone development because knockout results in lack of a mineralized skeleton. The proximal Osx promoter contains numerous binding sequences for MyoD and 14 repeats of a binding sequence for Myf5. These basic helix-loop-helix (bHLH) transcription factors have a critical role in MB differentiation and muscle development. We tested the hypothesis that bHLH transcription factors also support OB differentiation through regulation of Osx. Transfection of a MyoD expression vector into two primitive mesenchymal cell lines, C3H/10T1/2 and C2C12, stimulated a 1.2-kb Osx promoter-luciferase reporter 70-fold. Myf5 stimulated the Osx promoter 6-fold. Deletion analysis of the promoter revealed that one of three proximal bHLH sites is essential for MyoD activity. The Myf5 repeat conferred 60% of Myf5 activity with additional upstream sequence required for full activity. MyoD bound the active bHLH sequence and its 3'-flanking region, as shown by EMSA and chromatin immunoprecipitation assays. Real-time PCR revealed that primitive C2C12 and C3H/10T1/2 cells, pre-osteoblastic MC3T3 cells, and undifferentiated primary marrow stromal cells express the muscle transcription factors. C2C12 cells, which differentiate to MB spontaneously and form myotubules, were treated with bone morphogenetic protein 2 (BMP-2) to induce OB differentiation. BMP-2 stimulated expression of Osx and the differentiation marker alkaline phosphatase and blocked myotubule development. BMP-2 suppressed the muscle transcription factor myogenin, but expression of MyoD and Myf5 persisted. Silencing of MyoD inhibited BMP-2 stimulation of Osx and blocked the later appearance of bone alkaline phosphatase. MyoD support of Osx transcription contributes to early OB differentiation.

Transcriptional regulation of the osterix (Osx, Sp7) promoter by tumor necrosis factor identifies disparate effects of mitogen-activated protein kinase and NF kappa B pathways.

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-alpha (TNF-alpha), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFkappaB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFkappaB nuclear entry. The forced expression of high levels of NFkappaB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

Medical School Performance, Alumni Membership, and Giving: How Do Scholarship Recipients and Non-Recipients Differ?

PURPOSE: This study examines student recipients of merit, need-based, service, or minority scholarships, their performance in medical school, and the relationship to future alumni association membership and financial giving. METHOD: Retrospective data on grade-point average attained across the four-year curriculum and extracurricular activities reported at graduation were collected on students at the University of Kentucky College of Medicine from 1981-1991. Comparisons of academic performance and participation in institutional activities were made across scholarship recipients and non-recipients. These data were then linked to other data tracking alumni association membership and institutional giving. RESULTS: Compared to other scholarship recipients and non-recipients, merit scholars were more likely to be ranked above their class medians and be involved in extracurricular activities, including membership in Alpha Omega Alpha. However, seven years post-graduation, there was no difference between scholarship recipients and non-recipients in alumni association membership or donations to the medical school. Instead, students graduating in the upper half of their class, as compared to graduates in the lower half, and UKCOM graduates who attended the University of Kentucky as undergraduates, rather than students who attended other in-state or out-of state institutions, were more likely to join the medical alumni association. Alumni association members were more likely than non-members to make donations to the institution. CONCLUSIONS: More should be done to ensure that graduates who received scholarships are afforded meaningful ways to give back to the institution that supported them as students.

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis.

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

A comparison of applicant and matriculant trends, and rising costs of medical education in United States medical schools and at the University of Kentucky College of Medicine.

This paper addresses fluctuations in the applicant and matriculant pools both across United States medical schools and at the University of Kentucky College of Medicine (UKCOM) for 1992-2002. It also presents data regarding the increasing costs of a medical education. Over the past decade, both nationally and at the UKCOM, there has been an over-all reduction in the number of applicants to medical school. In this changing applicant pool, the percentage of female matriculants has increased both nationally and at the UKCOM. However, the number of underrepresented minorities applying to and matriculating in the US and at the UKCOM has dropped since the mid-1990s. Although the applicant pool has decreased in size over the time period examined, the academic quality of applicants as measured by the undergraduate grade point average and Medical College Admission Test scores has increased both nationally and at UKCOM. Costs of a medical education have risen over time, as has the debt burden of medical school graduates due to increasing undergraduate debt, consumer debt, and medical school tuition. Potential causes for and implication of these changing trends are discussed.

Expression of the osteoblast differentiation factor RUNX2 (Cbfa1/AML3/Pebp2alpha A) is inhibited by tumor necrosis factor-alpha.

The transcription factor RUNX2 (Cbfa1/AML3/Pebp2alphaA) is a critical regulator of osteoblast differentiation. We investigated the effect of the inflammatory cytokine tumor necrosis factor alpha (TNF) on the expression of RUNX2 because TNF is known to inhibit differentiation of osteoblasts from pluripotent progenitor cells. TNF treatment of fetal calvaria precursor cells or MC3T3-E1 clonal pre-osteoblastic cells caused a dose-dependent suppression of RUNX2 steady state mRNA as measured by reverse transcription-PCR. The IC(50) for TNF inhibition was 0.6 ng/ml. TNF suppression of RUNX2 mRNA was confirmed using Northern analysis. The effect of TNF was studied using isoform-specific primers that flanked unique regions of two major RUNX2 isoforms. TNF suppressed expression of the mRNA coding for the shorter MRIPV isoform by >90% while inhibiting expression of the mRNA for the longer MASNS isoform by 50%. RUNX2 nuclear content was evaluated by electrophoretic mobility shift assay using a rat osteocalcin promoter binding sequence as probe and by Western analysis. TNF reduced nuclear RUNX2 protein. Inhibition of new protein synthesis with cycloheximide failed to prevent TNF inhibition of RUNX2 mRNA, suggesting that a newly translated protein did not mediate the TNF effect. RUNX2 mRNA half-life was 1.8 h and reduced to 0.9 h by TNF. The effect of TNF on RUNX2 gene transcription was evaluated using a 0.6-kb RUNX2 promoter-luciferase reporter in MC3T3-E1 cells. TNF caused a dose-dependent inhibition of transcription to 50% of control values. The inhibitory effect of TNF was preserved with deletions to nucleotide -108 upstream of the translational start site; however, localization downstream of nucleotide -108 was obscured by loss of basal activity. Our results indicate that TNF regulates RUNX2 expression at multiple levels including destabilization of mRNA and suppression of transcription. The disproportionate inhibition of RUNX2 nuclear protein suggests that additional post-transcriptional mechanisms may be occurring. Suppression of RUNX2 by TNF may decrease osteoblast differentiation and inhibit bone formation in TNF excess states.