Development of the internal family systems model: Honoring contributions from family systems therapies.

We describe Richard Schwartz's development of the Internal Family Systems model (IFS) from his position as a Structural/Strategic family therapist. Four decades ago, Schwartz struggled to help clients who exhibited serious risk of harm to self and others. Through a process of inquiry, he began to work with the positive intentions behind his most challenging clients' harmful thoughts and behaviors. He applied foundational ideas from family systems thinking to patterns of internal experiences. As he experimented with ways of applying these ideas, he created an approach to healing. We summarize the IFS model delineating ways a range of family systems theory and practice inform its development and contribute to its best practice. Our purposes are to inform IFS practitioners who are not trained in foundational family systems models as well as to acknowledge the significant contributions family therapy theories made in the development and best practice of the IFS model.

The Association between Sex Hormones, Pubertal Milestones and Benzophenone-3 Exposure, Measured by Urinary Biomarker or Questionnaire.

Experimental studies have suggested benzophenone-3 (BP-3), a sunscreen ingredient, may have endocrine-disrupting properties. A cohort of girls were recruited at ages 6-7 years and returned semi-annually for pubertal maturation staging, provided blood for serum hormone analyses [estradiol, estrone, testosterone, dehydroepiandrosterone-sulfate (DHEA-S)], and urine to measure BP-3 concentrations. We found a significant negative linear association between amount of reported sunscreen use and testosterone levels at the onset of puberty (N = 157, adjusted ß = -0.0163, 97.5% CI:-0.0300,-0.0026). The 2nd quartile of the BP-3 biomarker had earlier thelarche compared to the 1st quartile (N = 282, adjusted HR = 1.584, 97.5% CI:1.038,2.415). Results suggest that higher report of sunscreen use may be associated with lower testosterone levels at thelarche and a non-linear relationship between the BP-3 urinary biomarker and onset of puberty, although the clinical significance of the finding is limited and may be a random effect. Improved methods of BP-3 exposure characterization are needed.

CD4 Expression and Env Conformation Are Critical for HIV-1 Restriction by SERINC5.

Serine incorporator 5 (SERINC5) is a recently identified restriction factor that strongly blocks HIV-1 entry but is counteracted by Nef. Notably, tier 1 HIV-1 Env proteins are sensitive to SERINC5, whereas the majority of tier 2/3 Env proteins are resistant to SERINC5, when viruses are produced from CD4-negative cells and tested by a single-round replication assay. Here, we investigated the Env-dependent SERINC5 antiviral mechanism by comparing tier 1 NL Env with tier 3 AD8 Env proteins. We found that when NL and AD8 viruses were inoculated into CD4+ T cells and human peripheral blood mononuclear cells (PBMCs), the propagation of the two viruses was restricted to a similar level when Nef was not expressed. Using a bimolecular fluorescence complementation (BiFC) assay, we detected Env-Env association and Env-SERINC5 interactions. A much greater level of NL Env-SERINC5 interactions was detected than was AD8 Env-SERINC5 interactions, which was further validated by immunoprecipitation assays. In addition, SERINC5 dissociated the NL Env trimeric complex more effectively than the AD8 Env trimeric complex when CD4 was not expressed. However, when CD4 was expressed, SERINC5 became more capable of interacting with AD8 Env and dissociating its trimeric complex. Moreover, AD8 and several other tier 2/3 viruses produced in the presence of CD4 became sensitive to SERINC5 when measured by the single-round replication assay. Because tier 1 and tier 2/3 Env trimers have open and closed conformations, respectively, and CD4 opens the closed conformation, we conclude that SERINC5 selectively dissociates Env trimers with an open conformation to restrict HIV-1 replication.IMPORTANCE Restriction factors provide the first line of defense against retrovirus infection by posing several blocks to the viral replication cycle. SERINC5 is a novel restriction factor that strongly blocks HIV-1 entry, although it is counteracted by Nef. Currently, it is still unclear how HIV-1 entry is blocked by SERINC5. Notably, this entry block is dependent on viral Env proteins. Laboratory-adapted HIV-1 strains are sensitive, whereas primary isolates are highly resistant to SERINC5. Env proteins mediate virus entry via extensive conformational rearrangements from a closed ground state to a CD4-bound open state. We detected Env-Env associations and Env-SERINC5 interactions in live cells by a novel bimolecular fluorescence assay. We demonstrate that CD4 expression increases the Env sensitivity to SERINC5 and allows SERINC5 to dissociate the Env complex, suggesting that SERINC5 restriction is dependent on Env conformation. Our results provide new insights into the poorly defined Env-dependent SERINC5 antiviral mechanism.

Innate Sensing of Influenza A Virus Hemagglutinin Glycoproteins by the Host Endoplasmic Reticulum (ER) Stress Pathway Triggers a Potent Antiviral Response via ER-Associated Protein Degradation.

Innate immunity provides an immediate defense against infection after host cells sense danger signals from microbes. Endoplasmic reticulum (ER) stress arises from accumulation of misfolded/unfolded proteins when protein load overwhelms the ER folding capacity, which activates the unfolded protein response (UPR) to restore ER homeostasis. Here, we show that a mechanism for antiviral innate immunity is triggered after the ER stress pathway senses viral glycoproteins. When hemagglutinin (HA) glycoproteins from influenza A virus (IAV) are expressed in cells, ER stress is induced, resulting in rapid HA degradation via proteasomes. The ER-associated protein degradation (ERAD) pathway, an important UPR function for destruction of aberrant proteins, mediates HA degradation. Three class I α-mannosidases were identified to play a critical role in the degradation process, including EDEM1, EDEM2, and ERManI. HA degradation requires either ERManI enzymatic activity or EDEM1/EDEM2 enzymatic activity when ERManI is not expressed, indicating that demannosylation is a critical step for HA degradation. Silencing of EDEM1, EDEM2, and ERManI strongly increases HA expression and promotes IAV replication. Thus, the ER stress pathway senses influenza HA as "nonself" or misfolded protein and sorts HA to ERAD for degradation, resulting in inhibition of IAV replication.IMPORTANCE Viral nucleic acids are recognized as important inducers of innate antiviral immune responses that are sensed by multiple classes of sensors, but other inducers and sensors of viral innate immunity need to be identified and characterized. Here, we used IAV to investigate how host innate immunity is activated. We found that IAV HA glycoproteins induce ER stress, resulting in HA degradation via ERAD and consequent inhibition of IAV replication. In addition, we have identified three class I α-mannosidases, EDEM1, EDEM2, and ERManI, which play a critical role in initiating HA degradation. Knockdown of these proteins substantially increases HA expression and IAV replication. The enzymatic activities and joint actions of these mannosidases are required for this antiviral activity. Our results suggest that viral glycoproteins induce a strong innate antiviral response through activating the ER stress pathway during viral infection.

Puberty-specific promotion of mammary tumorigenesis by a high animal fat diet.

INTRODUCTION: Increased animal fat consumption is associated with increased premenopausal breast cancer risk in normal weight, but not overweight, women. This agrees with our previous findings in obesity-resistant BALB/c mice, in which exposure to a high saturated animal fat diet (HFD) from peripuberty through adulthood promoted mammary tumorigenesis. Epidemiologic and animal studies support the importance of puberty as a life stage when diet and environmental exposures affect adult breast cancer risk. In this study, we identified the effects of peripubertal exposure to HFD and investigated its mechanism of enhancing tumorigenesis. METHODS: Three-week-old BALB/c mice fed a low-fat diet (LFD) or HFD were subjected to 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis. At 9 weeks of age, half the mice on LFD were switched to HFD (LFD-HFD group) and half the mice on HFD were switched to LFD (HFD-LFD group). Tumor gene expression was evaluated in association with diet and tumor latency. RESULTS: The peripubertal HFD reduced the latency of DMBA-induced mammary tumors and was associated with tumor characteristics similar to those in mice fed a continuous HFD. Notably, short-latency tumors in both groups shared gene expression characteristics and were more likely to have adenosquamous histology. Both HFD-LFD and continuous HFD tumors showed similar gene expression patterns and early latency. Adult switch from HFD to LFD did not reverse peripubertal HFD tumor promotion. Increased proliferation, hyperplasia, and macrophages were present in mammary glands before tumor development, implicating these as possible effectors of tumor promotion. Despite a significant interaction between pubertal diet and carcinogens in tumor promotion, peripubertal HFD by itself produced persistent macrophage recruitment to mammary glands. CONCLUSIONS: In obesity-resistant mice, peripubertal HFD is sufficient to irreversibly promote carcinogen-induced tumorigenesis. Increased macrophage recruitment is likely a contributing factor. These results underscore the importance of early life exposures to increased adult cancer risk and are consistent with findings that an HFD in normal weight premenopausal women leads to increased breast cancer risk. Notably, short-latency tumors occurring after peripubertal HFD had characteristics similar to human basal-like breast cancers that predominantly develop in younger women.

Benzophenone-3 alters expression of genes encoding vascularization and epithelial-mesenchymal transition functions during Trp53-null mammary tumorigenesis.

Benzophenone-3 (also referred to as oxybenzone) is a putative endocrine disrupting chemical and common ingredient in sunscreens and other personal care products. We previously showed that benzophenone-3 was promotional for epithelial tumorigenesis in mice fed adult high-fat diet, while protective against the incidence of more aggressive spindle cell tumors in the same treatment group. In this study, we show that benzophenone-3 reduces epithelial to mesenchymal transition in the epithelial tumors of these mice. This reduction in epithelial to mesenchymal transition is associated with altered expression of several genes involved in regulation of angiogenesis and epithelial to mesenchymal transition. Among the genes altered in expression, Timp1 is of particular interest because benzophenone-3 suppressed both migration and Timp1 expression in a mammary tumor cell line that displays epithelial to mesenchymal transition characteristics. These alterations in gene expression plausibly stabilize the vasculature of epithelial carcinomas and contribute to benzophenone-3 promotion of epithelial tumors, while at the same time suppress epithelial to mesenchymal transition and suppress incidence of spindle cell tumors.

Amphiregulin mediates progesterone-induced mammary ductal development during puberty.

INTRODUCTION: Puberty is a period of increased susceptibility to factors that cause increased breast cancer risk in adulthood. Mammary end buds (EBs) that develop during puberty are believed to be the targets of breast cancer initiation. Whereas the role of estrogen (E) has been extensively studied in pubertal mammary gland development, the role of progesterone (P) during puberty is less defined. METHODS: Pubertal and prepubertal ovariectomized mice were treated with vehicle control (C), E, P, or E+P. Mammary glands from these mice were analyzed for changes in morphology, proliferation, and expression of the downstream targets amphiregulin (AREG) and receptor activator of NF-κB ligand (RANKL). RESULTS: P, acting specifically through the progesterone receptor, induced increases in mammary gland proliferation and EB formation that were associated with increased AREG expression in ducts and EBs. E, acting specifically through the estrogen receptor, produced similar responses also mediated by AREG. Blocking AREG action by treatment with an EGFR inhibitor completely abrogated the effect of P on EB formation and proliferation and significantly reduced proliferation within ducts. P also increased expression of RANKL, primarily in ducts. Treatment with RANK-Fc, an inhibitor of RANKL, reduced P-dependent proliferation in ducts and to a lesser extent in EB, but did not cause EB regression. CONCLUSIONS: These results demonstrate a novel P-specific effect through AREG to cause EB formation and proliferation in the developing mammary gland both before and during puberty. Thus, hormones and/or factors in addition to E that upregulate AREG can promote mammary gland development and have the potential to affect breast cancer risk associated with pubertal mammary gland development.

Pubertal high fat diet: effects on mammary cancer development.

INTRODUCTION: Epidemiological studies linking dietary fat intake and obesity to breast cancer risk have produced inconsistent results. This may be due to the difficulty of dissociating fat intake from obesity, and/or the lack of defined periods of exposure in these studies. The pubertal mammary gland is highly sensitive to cancer-causing agents. We assessed how high fat diet (HFD) affects inflammation, proliferative, and developmental events in the pubertal gland, since dysregulation of these can promote mammary tumorigenesis. To test the effect of HFD initiated during puberty on tumorigenesis, we utilized BALB/c mice, for which HFD neither induces obesity nor metabolic syndrome, allowing dissociation of HFD effects from other conditions associated with HFD. METHODS: Pubertal BALB/c mice were fed a low fat diet (12% kcal fat) or a HFD (60% kcal fat), and subjected to carcinogen 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumorigenesis. RESULTS: HFD elevated mammary gland expression of inflammatory and growth factor genes at 3 and 4 weeks of diet. Receptor activator of nuclear factor kappa-B ligand (RANKL), robustly induced at 4 weeks, has direct mitogenic activity in mammary epithelial cells and, as a potent inducer of NF-κB activity, may induce inflammatory genes. Three weeks of HFD induced a transient influx of eosinophils into the mammary gland, consistent with elevated inflammatory factors. At 10 weeks, prior to the appearance of palpable tumors, there were increased numbers of abnormal mammary epithelial lesions, enhanced cellular proliferation, increased growth factors, chemokines associated with immune-suppressive regulatory T cells, increased vascularization, and elevated M2 macrophages. HFD dramatically reduced tumor latency. Early developing tumors were more proliferative and were associated with increased levels of tumor-related growth factors, including increased plasma levels of HGF in tumor-bearing animals. Early HFD tumors also had increased vascularization, and more intra-tumor and stromal M2 macrophages. CONCLUSIONS: Taken together in this non-obesogenic context, HFD promotion of inflammatory processes, as well as local and systemically increased growth factor expression, are likely responsible for the enhanced tumorigenesis. It is noteworthy that although DMBA mutagenesis is virtually random in its targeting of genes in tumorigenesis, the short latency tumors arising in animals on HFD showed a unique gene expression profile, highlighting the potent overarching influence of HFD.

Moving from acceptance toward transformation with Internal Family Systems Therapy (IFS).

Clients come to psychotherapy intent on changing, rather than accepting, their unwanted behaviors, emotions, or thoughts. The problem often is, however, that their lack of self-acceptance is the primary obstacle to change. This article describes how one approach, the Internal Family Systems (IFS) model, fosters clients' acceptance of all parts of themselves no matter how destructive, and how that acceptance can lead to the transformation of those parts and, in turn, of other people.

Benzophenone-3 exposure alters composition of tumor infiltrating immune cells and increases lung seeding of 4T1 breast cancer cells.

Environmental chemicals are a persistent and pervasive part of everyday life. A subset of environmental chemicals are xenoestrogens, compounds that bind to the estrogen receptor (ER) and drive estrogen-related processes. One such chemical, benzophenone-3 (BP3), is a common chemical in sunscreen. It is a potent UV protectant but also is quickly absorbed through the skin. While it has been approved by the FDA, there is a renewed interest in the safety of BP3, particularly in relation to breast cancer. The focus of this study was to examine the impact that BP3 has on triple negative breast cancer (TNBC) through alterations to cells in the immune microenvironment. In this study, we exposed female mice to one of two doses of BP3 before injecting them with a TNBC cell line. Several immune endpoints were examined both in the primary tissues and from in vitro studies of T cell behavior. Our studies revealed that in the lung tumor microenvironment, exposure to BP3 not only increased the number of metastases, but also the total area of tumor coverage. We also found that BP3 caused alterations in immune populations in a tissue-dependent manner, particularly in T cells. Taken together, our data suggest that while BP3 may not directly affect the proliferation of TNBC, growth and metastasis of TNBC-derived tumors can be altered by BP3 exposures via the alterations in the immune populations of the tumor microenvironment.

RNF185 regulates proteostasis in Ebolavirus infection by crosstalk between the calnexin cycle, ERAD, and reticulophagy.

Virus infection affects cellular proteostasis and provides an opportunity to study this cellular process under perturbation. The proteostasis network in the endoplasmic reticulum (ER) is composed of the calnexin cycle, and the two protein degradation pathways ER-associated protein degradation (ERAD) and ER-to-lysosome-associated degradation (ERLAD/ER-phagy/reticulophagy). Here we show that calnexin and calreticulin trigger Zaire Ebolavirus (EBOV) glycoprotein GP1,2 misfolding. Misfolded EBOV-GP1,2 is targeted by ERAD machinery, but this results in lysosomal instead of proteasomal degradation. Moreover, the ER Ub ligase RNF185, usually associated with ERAD, polyubiquitinates EBOV-GP1,2 on lysine 673 via ubiquitin K27-linkage. Polyubiquinated GP1,2 is subsequently recruited into autophagosomes by the soluble autophagy receptor sequestosome 1 (SQSTM1/p62), in an ATG3- and ATG5-dependent manner. We conclude that EBOV hijacks all three proteostasis mechanisms in the ER to downregulate GP1,2 via polyubiquitination and show that this increases viral fitness. This study identifies linkages among proteostasis network components previously thought to function independently.

Toward a digital analysis of environmental impacts on rodent mammary gland density during critical developmental windows.

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies.

Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection.Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: ß-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A1; BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

CCAAT/enhancer binding protein ß-deficiency enhances type 1 diabetic bone phenotype by increasing marrow adiposity and bone resorption.

Bone loss in type 1 diabetes is accompanied by increased marrow fat, which could directly reduce osteoblast activity or result from altered bone marrow mesenchymal cell lineage selection (adipocyte vs. osteoblast). CCAAT/enhancer binding protein beta (C/EBPß) is an important regulator of both adipocyte and osteoblast differentiation. C/EBPß-null mice have delayed bone formation and defective lipid accumulation in brown adipose tissue. To examine the balance of C/EBPß functions in the diabetic context, we induced type 1 diabetes in C/EBPß-null (knockout, KO) mice. We found that C/EBPß deficiency actually enhanced the diabetic bone phenotype. While KO mice had reduced peripheral fat mass compared with wild-type mice, they had 5-fold more marrow adipocytes than diabetic wild-type mice. The enhanced marrow adiposity may be attributed to compensation by C/EBPδ, peroxisome proliferator-activated receptor-γ2, and C/EBPα. Concurrently, we observed reduced bone density. Relative to genotype controls, trabecular bone volume fraction loss was escalated in diabetic KO mice (-48%) compared with changes in diabetic wild-type mice (-22%). Despite greater bone loss, osteoblast markers were not further suppressed in diabetic KO mice. Instead, osteoclast markers were increased in the KO diabetic mice. Thus, C/EBPß deficiency increases diabetes-induced bone marrow (not peripheral) adipose depot mass, and promotes additional bone loss through stimulating bone resorption. C/EBPß-deficiency also reduced bone stiffness and diabetes exacerbated this (two-way ANOVA P < 0.02). We conclude that C/EBPß alone is not responsible for the bone vs. fat phenotype switch observed in T1 diabetes and that suppression of CEBPß levels may further bone loss and decrease bone stiffness by increasing bone resorption.

Selective inhibitors of Kv11.1 regulate IL-6 expression by macrophages in response to TLR/IL-1R ligands.

The mechanism by which the platelet-endothelial cell adhesion molecule PECAM-1 regulates leukodiapedesis, vascular endothelial integrity, and proinflammatory cytokine expression in vivo is not known. We recently identified PECAM-1 as a negative regulator of Kv11.1, a specific voltage-gated potassium channel that functioned in human macrophages to reset a resting membrane potential following depolarization. We demonstrate here that dofetilide (DOF), a selective inhibitor of the Kv11.1 current, had a profound inhibitory effect on neutrophil recruitment in mice following TLR/IL-1R-elicited peritonitis or intrascrotal injection of IL-1 Beta, but had no effect on responses seen with TNF alpha. Furthermore, inhibitors of Kv11.1 (DOF, E4031, and astemizole), but not Kv1.3 (margatoxin), suppressed the expression of IL-6 and MCP-1 cytokines by murine resident peritoneal macrophages, while again having no effect on TNF alpha. In contrast, IL-6 expression by peritoneal mesothelial cells was unaffected. Using murine P388 cells, which lack endogenous C/EBP Beta expression and are unresponsive to LPS for the expression of both IL-6 and MCP-1, we observed that DOF inhibited LPS-induced expression of IL-6 mRNA following ectopic expression of wild-type C/EBP Beta, but not a serine-64 point mutant. Finally, DOF inhibited the constitutive activation of cdk2 in murine peritoneal macrophages; cdk2 is known to phosphorylate C/EBP Beta at serine-64. Taken together, our results implicate a potential role for Kv11.1 in regulating cdk2 and C/EBP Beta activity, where robust transactivation of both IL-6 and MCP-1 transcription is known to be dependent on serine-64 of C/EBP Beta. Our data might also explain the altered phenotypes displayed by PECAM-1 knockout mice in several disease models.

Benzophenone-3 promotion of mammary tumorigenesis is diet-dependent.

Benzophenone-3 is a putative endocrine disrupting chemical and common ingredient in sunscreens. The potential of endocrine disrupting chemicals to act as agonists or antagonists in critical hormonally regulated processes, such as mammary gland development and mammary tumorigenesis, demands evaluation of its potential in promoting breast cancer. This study identifies the effects of BP-3 on mammary tumorigenesis with high-fat diet during puberty versus adulthood in Trp53-null transplant BALB/c mice. Benzophenone-3 exposure yielded levels in urine similar to humans subjected to heavy topical sunscreen exposure. Benzophenone-3 was protective for epithelial tumorigenesis in mice fed lifelong low-fat diet, while promotional for epithelial tumorigenesis in mice fed adult high-fat diet. Benzophenone-3 increased tumor cell proliferation, decreased tumor cell apoptosis, and increased tumor vascularity dependent on specific dietary regimen and tumor histopathology. Even in instances of an ostensibly protective effect, other parameters suggest greater risk. Although benzophenone-3 seemed protective on low-fat diet, spindle cell tumors arising in these mice showed increased proliferation and decreased apoptosis. This points to a need for further studies of benzophenone-3 in both animal models and humans as a potential breast cancer risk factor, as well as a more general need to evaluate endocrine disrupting chemicals in varying dietary contexts.

Bone inflammation and altered gene expression with type I diabetes early onset.

Type I diabetes is associated with bone loss and marrow adiposity. To identify early events involved in the etiology of diabetic bone loss, diabetes was induced in mice by multiple low dose streptozotocin injections. Serum markers of bone metabolism and inflammation as well as tibial gene expression were examined between 1 and 17 days post-injection (dpi). At 3 dpi, when blood glucose levels were significantly elevated, body, fat pad and muscle mass were decreased. Serum markers of bone resorption and formation significantly decreased at 5 dpi in diabetic mice and remained suppressed throughout the time course. An osteoclast gene, TRAP5 mRNA, was suppressed at early and late time points. Suppression of osteogenic genes (runx2 and osteocalcin) and induction of adipogenic genes (PPARgamma2 and aP2) were evident as early as 5 dpi. These changes were associated with an elevation of serum cytokines, but more importantly we observed an increase in the expression of cytokines in bone, supporting the idea that bone, itself, exhibits an inflammatory response during diabetes induction. This inflammation could in turn contribute to diabetic bone pathology. IFN-gamma (one of the key cytokines elevated in bone and known to be involved in bone regulation) deficiency did not prevent diabetic bone pathology. Taken together, our findings indicate that bone becomes inflamed with the onset of T1-diabetes and during this time bone phenotype markers become altered. However, inhibition of one cytokine, IFN-gamma was not sufficient to prevent the rapid bone phenotype changes.

Differential roles of C/EBP beta regulatory domains in specifying MCP-1 and IL-6 transcription.

C/EBPbeta is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. To examine the roles of the C/EBPbeta transactivation and regulatory domains in LPS-induced MCP-1 and IL-6 expression, we expressed various N-terminal truncations and deletions of C/EBPbeta in P388 murine B lymphoblasts, which lack endogenous C/EBPbeta expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. Unexpectedly, a region between amino acids 105 and 212 of C/EBPbeta that includes regulatory domains 1 and 2 facilitates C/EBPbeta activation of IL-6 expression, while having an inhibitory effect on MCP-1 expression. Thus, this region can mediate promoter-specific effects on cytokine and chemokine gene transcription. LIP, the naturally occurring truncated form of C/EBPbeta, largely retains these regulatory domains and stimulates IL-6 but not MCP-1 transcription.

The Proliferative Response to p27 Down-Regulation in Estrogen Plus Progestin Hormonal Therapy is Lost in Breast Tumors.

Increased proliferation and breast cancer risk has been observed in postmenopausal women receiving estrogen (E) + progestin hormone replacement therapy (HRT). Progestin action is mediated through two progesterone receptor (PR) isoforms, PRA and PRB, with unique transcriptional activity and function. The current study examines hormonal regulation of PR isoforms in the normal postmenopausal human breast and the mechanism by which progestins increase proliferation and breast cancer risk. Archival benign breast biopsies from postmenopausal and premenopausal women, and luminal breast tumor biopsies from postmenopausal women, were analyzed for regulation of PRA and PRB expression by E and E+medroxyprogesterone acetate (MPA). In the postmenopausal breast without HRT, PRA and PRB expression was decreased compared to the premenopausal breast. Both E (n = 12) and E+MPA (n = 13) HRT in the postmenopausal breast were associated with increased PRA and PRB expression, increased nuclear cyclin E expression, and decreased nuclear p27 expression compared to no HRT (n = 16). With E+MPA HRT, there was a further decrease in nuclear p27 and increased Receptor Activator of NF-kappa B Ligand (RANKL) expression compared to E-alone HRT. In luminal breast cancers, E+MPA HRT (n = 6) was also associated with decreased nuclear expression of the cell cycle inhibitor p27 compared to E HRT (n = 6), but was not associated with increased proliferation. These results suggest that p27 mediates progestin-induced proliferation in the normal human breast and that regulation of this proliferative response by E+MPA is lost in breast tumors.

C/EBPß LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

CCAAT/enhancer binding protein ß (C/EBPß) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPß regulation of PR expression. All three C/EBPß isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPß and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPß isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPß and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPß and PRB largely co-localized. We suggest a critical role for C/EBPß, particularly LIP, in PRB expression.