Inhibitor of differentiation-4 (Id4) stimulates pigmentation in melanoma leading to histiocyte infiltration.

TGF-ß and the inhibitors of differentiation (Id) are linked. Smad7 and other TGF-ß inhibitors can potently suppress melanomagenesis; however, little work examining Ids has been reported in melanoma, particularly for Id4. Here, we report that Id4, but not Id2 or Id3 expression, surprisingly, activated robust melanin production in xenografts of previously amelanotic (lacking pigment) 1205Lu/Smad7 (S7) cells. Fontana-Masson stain and de-novo expression of MART-1 and tyrosinase proteins confirmed melanin production. Additionally, pigment-laden CD163+ mouse histiocytes with areas of extensive necrosis were found throughout S7/Id4 tumors, but not in parental 1205Lu, S7/Id2 or S7Id3-derived tumors. Mechanistic investigation revealed increased nuclear M-microphthalmia-associated transcription factor (MITF) and MART-1 promoter activation following Id4 expression in 1205Lu and WM852 melanoma cells, suggesting broader implications for Id4 in melanin synthesis. In human tumors, melanin colocalized with Id4 expression establishing a correlation. Current chemotherapeutics for melanoma are only marginally effective. Immunotherapy provides the most promise, yet the role of innate immunity is poorly understood. Here, TGF-ß suppression followed by Id4 expression results in extensive melanin synthesis and robust histiocyte recruitment following tumorigenesis, a novel role for Id4. Our results suggest that TGF-ß suppression coupled with pigment overproduction triggers an innate immune response resulting in tumor necrosis.

Incentives to create and sustain healthy behaviors: technology solutions and research needs.

Health-related technology, its relevance, and its availability are rapidly evolving. Technology offers great potential to minimize and/or mitigate barriers associated with achieving optimal health, performance, and readiness. In support of the U.S. Army Surgeon General's vision for a "System for Health" and its Performance Triad initiative, the U.S. Army Telemedicine and Advanced Technology Research Center hosted a workshop in April 2013 titled "Incentives to Create and Sustain Change for Health." Members of government and academia participated to identify and define the opportunities, gain clarity in leading practices and research gaps, and articulate the characteristics of future technology solutions to create and sustain real change in the health of individuals, the Army, and the nation. The key factors discussed included (1) public health messaging, (2) changing health habits and the environmental influence on health, (3) goal setting and tracking, (4) the role of incentives in behavior change intervention, and (5) the role of peer and social networks in change. This report summarizes the recommendations on how technology solutions could be employed to leverage evidence-based best practices and identifies gaps in research where further investigation is needed.

Leveraging technology: creating and sustaining changes for health.

OBJECTIVE: The rapid growth and evolution of health-related technology capabilities are driving an established presence in the marketplace and are opening up tremendous potential to minimize and/or mitigate barriers associated with achieving optimal health, performance, and readiness. This article summarizes technology-based strategies that promote healthy habits related to physical activity, nutrition, and sleep. MATERIALS AND METHODS: The Telemedicine and Advanced Technology Research Center convened a workshop titled "Leveraging Technology: Creating & Sustaining Changes for Health" (May 29-30, 2013, Fort Detrick, MD). Participants included experts from academia (n=3), government (n=33), and industry (n=16). A modified Delphi method was used to establish expert consensus in six topic areas: (1) physical activity, (2) nutrition, (3) sleep, (4) incentives for behavior change, (5) usability/interoperability, and (6) mobile health/open platform. RESULTS: Overall, 162 technology features, constructs, and best practices were reviewed and prioritized for physical activity monitors (n=29), nutrition monitors (n=35), sleep monitors (n=24), incentives for change (n=36), usability and interoperability (n=25), and open data (n=13). CONCLUSIONS: Leading practices, gaps, and research needs for technology-based strategies were identified and prioritized. This information can be used to provide a research and development road map for (1) leveraging technology to minimize barriers to enhancing health and (2) facilitating evidence-based techniques to create and sustain healthy behaviors.

Key enablers to facilitate healthy behavior change: workshop summary.

The increases in preventable chronic diseases and the rising costs of health care are unsustainable. The US Army Surgeon General's vision to transition from a health care system to a system of health requires the identification of key health enablers to facilitate the adoption of healthy behaviors. In support of this vision, the US Army Telemedicine and Advanced Technology Research Center hosted a workshop in April 2013 titled "Incentives to Create and Sustain Change for Health." Members of government and academia participated to identify key health enablers that could ultimately be leveraged by technology. The key health enablers discussed included (1) public health messaging, (2) changing health habits and the environmental influence on health, (3) goal setting and tracking, (4) the role of incentives in behavior-change intervention, and (5) the role of peer and social networks on change. This report summarizes leading evidence and the group consensus on evidence-based practices with respect to the key enablers in creating healthy behavior change.

Id2, Id3 and Id4 overcome a Smad7-mediated block in tumorigenesis, generating TGF-ß-independent melanoma.

The role for the inhibitors of differentiation (Ids) proteins in melanomagenesis has been poorly explored. In other cell types, Ids have been shown to contribute to cell proliferation, migration and angiogenesis and, along with a number of other genes, are direct downstream targets of the transforming growth factor (TGF)-ß pathway. Expression of Smad7, which suppress TGF-ß signaling, or synthetic TGF-ß inhibitors, was shown to potently suppress melanomagenesis. We found that endogenous Id2, Id3 and Id4 expression was elevated in 1205Lu versus 1205Lu cells constitutively expressing Smad7, indicating Ids may play a role in melanomagenesis. Therefore, the effects of Tet-inducible expression of Id2, Id3 or Id4 along with Smad7 in TGF-ß-dependent 1205Lu human melanoma cells were explored in vitro and in vivo. 1205Lu cells formed subcutaneous tumors in athymic mice, whereas cells expressing Smad7 failed to form tumors. However, 1205Lu cells expressing Smad7 along with doxycycline-induced Id2, Id3 or Id4 were able to overcome the potent tumorigenic block mediated by S7, to varying degrees. Conversely, Id small interfering RNA knockdown suppressed anchorage-independent growth of melanoma. Histology of tumors from 1205Lu cells expressing Smad7 + Id4 revealed an average of 31% necrosis, compared with 5.2% in tumors from 1205Lu with vector only. Downstream, Ids suppressed cyclin-dependent kinase inhibitors, and re-upregulated invasion and metastasis-related genes matrix metalloproteinase 2 (MMP2), MMP9, CXCR4 and osteopontin, shown previously to be downregulated in response to Smad7. This study shows that Id2, Id3 and Id4 are each able to overcome TGF-ß dependence, and establish a role for Ids as key mediators of TGF-ß melanomagenesis.

Ferrous iron is found in mesenteric lymph bound to TIMP-2 following hemorrhage/resuscitation.

Extracellular iron has been implicated in the pathogenesis of post-injury organ failure. However, the source(s) and biochemical species of this iron have not been identified. Based upon evidence that distant organ injury results from an increase in intestinal permeability, we looked for ferrous iron in mesenteric lymph in anesthetized rats undergoing hemorrhage and fluid resuscitation (H/R). Ferrous iron increased in lymph from 4.7 nmol/mg of protein prior to hemorrhage to 86.6 nmol/mg during resuscitation. Utilizing immuno-spin trapping in protein fractions that were rich in iron, we tentatively indentified protein carrier(s) of ferrous iron by MALDI-TOF MS. One of the identified proteins was the metalloproteinase (MMP) inhibitor, TIMP-2. Antibody to TIMP-2 immunoprecipitated 74% of the ferrozine detectable iron in its protein fraction. TIMP-2 binds iron in vitro at pH 6.3, which is typical of conditions in the mesentery during hemorrhage, but it retains the ability to inhibit the metalloproteases MMP-2 and MMP-9. In summary, there is a large increase in extracellular ferrous iron in the gut in H/R demonstrating dysregulation of iron homeostasis. We have identified, for the first time, the binding of extracellular iron to TIMP-2.

Smad7 restricts melanoma invasion by restoring N-cadherin expression and establishing heterotypic cell-cell interactions in vivo.

The list of transforming growth factor-beta (TGF-ß)-related proteins in non-canonical TGF-ß signaling is growing. Examples include receptor-Smads directing micro-RNA processing and inhibitory-Smads, e.g. Smad7, directing cell adhesion. Human skin grafts with fluorescently tagged melanoma cells revealed Smad7-expressing cells positioned themselves proximal to the dermal-epidermal junction and failed to form tumors, while control cells readily invaded and formed tumors within the dermis. Smad7 significantly inhibited ß-catenin T41/S45 phosphorylation associated with degradation and induced a 4.5-fold increase in full-length N-cadherin. Cell adhesion assays confirmed a strong interaction between Smad7-expressing cells and primary dermal fibroblasts mediated via N-cadherin, while control cells were incapable of such interaction. Immunofluorescent analysis of skin grafts indicated N-cadherin homotypic interaction at the surface of both Smad7 cells and primary dermal fibroblasts, in contrast to control melanoma cells. We propose that Smad7 suppresses ß-catenin degradation and promotes interaction with N-cadherin, stabilizing association with neighboring dermal fibroblasts, thus mitigating invasion.

VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells.

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 µM, VMY-1-103 increased both the proportion of cells in G(1) and p21(CIP1) protein levels. At higher concentrations (5 µM or 10 µM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 µM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop.

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis.

Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins of the retinoblastoma tumor suppressor family and is implicated in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. To investigate its proposed role in tumorigenesis, Id2 or deletion mutants were re-expressed in Id2(-/-) dermal fibroblasts. Ectopic expression of Id2 or mutants containing the central HLH domain increased S-phase cells, cell proliferation in low and normal serum and induced tumorigenesis when grafted or subcutaneously injected into athymic mice. Similar to their downregulation in human tumors, the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p15(INK4b) was decreased by Id2; the former by downregulation of its promoter by the Id2 HLH domain-mediated sequestration of E12/E47. Re-expression of p27(KIP1) in Id2-overexpressing cells reverted the hyperproliferative and tumorigenic phenotype, implicating Id2 as an oncogene working through p27(KIP1). These results tie together the previously observed misregulation of Id2 with a novel mechanism for tumorigenesis.

UVB upregulates the bax promoter in immortalized human keratinocytes via ROS induction of Id3.

Id3 belongs to the inhibitor of differentiation family of helix-loop-helix transcription factors, important in proliferation, differentiation and apoptosis. We showed that Id3, but not Id2 or Id1, mediates the UVB-sensitization of immortalized keratinocytes by inducing caspase 9-dependent apoptosis. In this study, quantitative PCR analysis revealed a time-dependent increase in Id3 mRNA induced by UVB, dependent on reactive oxygen species. UVB upregulated promoter activity of Id3, but not Id2, at early time points, as shown by reporter assays and also stabilized Id3 mRNA, increasing its half-life from 10 to approximately 60 min. We next examined downstream events related to UVB-induced Id3 upregulation and investigated the effects of UVB or ectopic expression of Id3 on bax promoter activity. Regulatory elements in the bax promoter that mediate transcriptional activation by UVB and Id3, in the absence of p53, were identified. Bax promoter deletion analysis revealed that transcriptional activation by UVB involves a 738-bp region upstream from the transcription start site of bax. Mimicking the effects of UVB, ectopic expression of Id3 also upregulated bax mRNA and activated this 738-bp fragment. Mutational analysis of the transcription binding sites further showed that point mutations of the E-box region found in the 738-bp fragment, but not in a 174-bp fragment, completely abolished Id3- and UVB-inducible bax promoter activity, thus confirming the importance of Id3 and UVB-mediated Id3 upregulation in activating the bax promoter. These results suggest a mechanism whereby reactive oxygen species upregulation of Id3 relieves repression of bax via E-box-binding factors.

Id2 protein is selectively upregulated by UVB in primary, but not in immortalized human keratinocytes and inhibits differentiation.

Solar ultraviolet B (UVB) acts as both an initiator and promoter in models of multistage skin carcinogenesis. We found that, whereas UVB induces apoptosis in human papillomavirus-16 E6/7-immortalized keratinocytes, it inhibits markers of differentiation in human foreskin keratinocytes (HFK). Potential mechanisms for this differential response were examined by DNA microarray, which revealed that UVB alters the expression of three of the four human inhibitor of differentiation/DNA binding (Id) proteins that comprise a class of helix-loop-helix family of transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. These results were verified by RT-PCR and immunoblot analysis of control and UVB-irradiated primary and immortalized keratinocytes. Whereas Id1 was downregulated in both cell types, Id2 expression was upregulated in primary HFK, but not immortalized cells. In contrast, Id3 expression was significantly increased only in immortalized cells. The differential expression pattern of Id2 in response to UVB was recapitulated in reporter constructs containing the 5' regulatory regions of this gene. Id2 promoter activity increased in response to UVB in HFK, but not in immortalized cells. To identify the regulatory elements in the Id2 promoter that mediate transcriptional activation by UVB in HFK, promoter deletion/mutation analysis was performed. Deletion analysis revealed that transactivation involves a 166 bp region immediately upstream to the Id2 transcriptional start site and is independent of c-Myc. The consensus E twenty-six (ETS) binding site at -120 appears to mediate UVB transcriptional activation of Id2 because point mutations at this site completely abrogated this response. Chromatin immunoprecipitation and electrophoretic mobility-shift assays verified that the Id2 promoter interacts with known Id2 promoter (ETS) binding factors Erg1/2 and Fli1, but not with c-Myc; and this interaction is enhanced after UVB exposure. Similar to the effects of UVB exposure, ectopic expression of Id2 protein in primary HFK resulted in inhibition of differentiation, as shown by decreased levels of the terminal differentiation marker keratin K1 and inhibition of involucrin crosslinking. Reduction of Id2 expression by small interfering RNAs attenuated the UVB-induced inhibition of differentiation in these cells. These results suggest that UVB-induced inhibition of differentiation of primary HFK is at least, in part, due to the upregulation of Id2, and that upregulation of Id2 by UVB might predispose keratinocytes to carcinogenesis by preventing their normal differentiation program.

Actin-based centripetal flow: phosphatase inhibition by calyculin-A alters flow pattern, actin organization, and actomyosin distribution.

Previous studies have suggested that the actin-based centripetal flow process in sea urchin coelomocytes is the result of a two-part mechanism, actin polymerization at the cell edge coupled with actomyosin contraction at the cell center. In the present study, we have extended the testing of this two-part model by attempting to stimulate actomyosin contraction via treatment of coelomocytes with the phosphatase inhibitor Calyculin A (CalyA). The effects of this drug were studied using digitally-enhanced video microscopy of living cells combined with immunofluorescent localization and scanning electron microscopy. Under the influence of CalyA, the coelomocyte actin cytoskeleton undergoes a radical reorganization from a dense network to one displaying an array of tangential arcs and radial rivulets in which actin and the Arp2/3 complex concentrate. In addition, the structure and dynamics of the cell center are transformed due to the accumulation of actin and membrane in this region and the constriction of the central actomyosin ring. Physiological evidence of an increase in actomyosin-based contractility following CalyA treatment was demonstrated in experiments in which cells generated tears in their cell centers in response to the drug. Western blotting and immunofluorescent localization with antibodies against the phosphorylated form of the myosin regulatory light chain (MRLC) suggested that the demonstrated constriction of actomyosin distribution was the result of CalyA-induced phosphorylation of MRLC. Overall, the results suggest that there is significant cross talk between the two underlying mechanisms of actin polymerization and actomyosin contraction, and indicate that changes in actomyosin tension may be translated into alterations in the structural organization of the actin cytoskeleton.

Wound closure in the lamellipodia of single cells: mediation by actin polymerization in the absence of an actomyosin purse string.

The actomyosin purse string is an evolutionarily conserved contractile structure that is involved in cytokinesis, morphogenesis, and wound healing. Recent studies suggested that an actomyosin purse string is crucial for the closure of wounds in single cells. In the present study, morphological and pharmacological methods were used to investigate the role of this structure in the closure of wounds in the peripheral cytoplasm of sea urchin coelomocytes. These discoidal shaped cells underwent a dramatic form of actin-based centripetal/retrograde flow and occasionally opened and closed spontaneous wounds in their lamellipodia. Fluorescent phalloidin staining indicated that a well defined fringe of actin filaments assembles from the margin of these holes, and drug studies with cytochalasin D and latrunculin A indicated that actin polymerization is required for wound closure. Additional evidence that actin polymerization is involved in wound closure was provided by the localization of components of the Arp2/3 complex to the wound margin. Significantly, myosin II immunolocalization demonstrated that it is not associated with wound margins despite being present in the perinuclear region. Pharmacological evidence for the lack of myosin II involvement in wound closure comes from experiments in which a microneedle was used to produce wounds in cells in which actomyosin contraction was inhibited by treatment with kinase inhibitors. Wounds produced in kinase inhibitor-treated cells closed in a manner similar to that seen with control cells. Taken together, our results suggest that an actomyosin purse string mechanism is not responsible for the closure of lamellar wounds in coelomocytes. We hypothesize that the wounds heal by means of a combination of the force produced by actin polymerization alone and centripetal flow. Interestingly, these cells did assemble an actomyosin structure around the margin of phagosome-like membrane invaginations, indicating that myosin is not simply excluded from the periphery by some general mechanism. The results indicate that the actomyosin purse string is not the only mechanism that can mediate wound closure in single cells.