Multiple roles for Bcl-3 in mammary gland branching, stromal collagen invasion, involution and tumor pathology.

BACKGROUND: The Bcl-3 protein is an atypical member of the inhibitor of -κB family that has dual roles as a transcriptional repressor and a coactivator for dimers of NF-κB p50 and p52. Bcl-3 is expressed in mammary adenocarcinomas and can promote tumorigenesis and survival signaling and has a key role in tumor metastasis. In this study, we have investigated the role of Bcl-3 in the normal mammary gland and impact on tumor pathology. METHODS: We utilized bcl-3-/- mice to study mammary gland structure in virgins and during gestation, lactation and early involution. Expression of involution-associated genes and proteins and putative Bcl-3 target genes was examined by qRT-PCR and immunoblot analysis. Cell autonomous branching morphogenesis and collagen I invasion properties of bcl-3-/- organoids were tested in 3D hydrogel cultures. The role of Bcl-3 in tumorigenesis and tumor pathology was also assessed using a stochastic carcinogen-induced mammary tumor model. RESULTS: Bcl-3-/- mammary glands demonstrated reduced branching complexity in virgin and pregnant mice. This defect was recapitulated in vitro where significant defects in bud formation were observed in bcl-3-/- mammary organoid cultures. Bcl-3-/- organoids showed a striking defect in protrusive collective fibrillary collagen I invasion associated with reduced expression of Fzd1 and Twist2. Virgin and pregnant bcl-3-/- glands showed increased apoptosis and rapid increases in lysosomal cell death and apoptosis after forced weaning compared to WT mice. Bcl-2 and Id3 are strongly induced in WT but not bcl-3-/- glands in early involution. Tumors in WT mice were predominately adenocarcinomas with NF-κB activation, while bcl-3-/- lesions were largely squamous lacking NF-κB and with low Bcl-2 expression. CONCLUSIONS: Collectively, our results demonstrate that Bcl-3 has a key function in mammary gland branching morphogenesis, in part by regulation of genes involved in extracellular matrix invasion. Markedly reduced levels of pro-survival proteins expression in bcl-3 null compared to WT glands 24 h post-weaning indicate that Bcl-3 has a role in moderating the rate of early phase involution. Lastly, a reduced incidence of bcl-3-/- mammary adenocarcinomas versus squamous lesions indicates that Bcl-3 supports the progression of epithelial but not metaplastic cancers.

VIVA1: a more invasive subclone of MDA-MB-134VI invasive lobular carcinoma cells with increased metastatic potential in xenograft models.

BACKGROUND: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. As few tools exist to study ILC metastasis, we isolated ILC cells with increased invasive properties to establish a spontaneously metastasising xenograft model. METHODS: MDA-MB-134VI ILC cells were placed in transwells for 7 days. Migrated cells were isolated and expanded to create the VIVA1 cell line. VIVA1 cells were compared to parental MDA-MB-134VI cells in vitro for ILC marker expression and relative proliferative and invasive ability. An intraductally injected orthotopic xenograft model was used to assess primary and metastatic tumour growth in vivo. RESULTS: Similar to MDA-MB-134VI, VIVA1 cells retained expression of oestrogen receptor (ER) and lacked expression of E-cadherin, however showed increased invasion in vitro. Following intraductal injection, VIVA1 and MDA-MB-134VI cells had similar primary tumour growth and survival kinetics. However, macrometastases were apparent in 7/10 VIVA1-injected animals. Cells from a primary orthotopic tumour (VIVA-LIG43) were isolated and showed similar proliferative rates but were also more invasive than parental cells. Upon re-injection intraductally, VIVA-LIG43 cells had more rapid tumour growth with similar metastatic incidence and location. CONCLUSIONS: We generated a new orthotopic spontaneously metastasising xenograft model for ER+ ILC amenable for the study of ILC metastasis.

Nuclear IMPDH Filaments in Human Gliomas.

The analysis of nuclear morphology plays an important role in glioma diagnosis and grading. We previously described intranuclear rods (rods) labeled with the SDL.3D10 monoclonal antibody against class III beta-tubulin (TUBB3) in human ependymomas. In a cohort of adult diffuse gliomas, we identified nuclear rods in 71.1% of IDH mutant lower-grade gliomas and 13.7% of IDH wild-type glioblastomas (GBMs). The presence of nuclear rods was associated with significantly longer postoperative survival in younger (≤65) GBM patients. Consistent with this, nuclear rods were mutually exclusive with Ki67 staining and their prevalence in cell nuclei inversely correlated with the Ki67 proliferation index. In addition, rod-containing nuclei showed a relative depletion of lamin B1, suggesting a possible association with senescence. To gain insight into their functional significance, we addressed their antigenic properties. Using a TUBB3-null mouse model, we demonstrate that the SDL.3D10 antibody does not bind TUBB3 in rods but recognizes an unknown antigen. In the present study, we show that rods show immunoreactivity for the nucleotide synthesizing enzymes inosine monophosphate dehydrogenase (IMPDH) and cytidine triphosphate synthetase. By analogy with the IMPDH filaments that have been described previously, we postulate that rods regulate the activity of nucleotide-synthesizing enzymes in the nucleus by sequestration, with important implications for glioma behavior.

Modulating SIRT1 activity variously affects thymic lymphoma development in mice.

SIRT1 is a protein deacetylase with a broad range of biological functions, many of which are known to be important in carcinogenesis, however much of the literature regarding the role of SIRT1 in cancer remains conflicting. In this study we assessed the effect of SIRT1 on the initiation and progression of thymic T cell lymphomas. We employed mouse strains in which SIRT1 activity was absent or could be reversibly modulated in conjunction with thymic lymphoma induction using either the N-nitroso-N-methylurea (NMU) carcinogenesis or the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) transgene. Decreased SIRT1 activity reduced the development of thymic lymphomas in the NMU-treated mice but was permissive for the formation of lung adenomas. Conversely, in the NPM-ALK transgenic mice, decreased SIRT1 activity had a modest promoting effect in the development of thymic lymphomas. The results of the work presented here add to the growing body of evidence that sirt1 is neither an outright oncogene nor a tumor suppressor. These opposing results in two models of the same disease suggest that the influence of sirt1 on carcinogenesis may lie in a role in tumor surveillance.

Ubiquitin COOH-terminal hydrolase L1 deletion is associated with urinary α-klotho deficiency and perturbed phosphate homeostasis.

Loss of ubiquitin COOH-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme required for neuronal function, led to hyperphosphatemia accompanied by phosphaturia in mice, while calcium homeostasis remained intact. We therefore investigated the mechanisms underlying the phosphate imbalance in Uchl1-/- mice. Interestingly, phosphaturia was not a result of lower renal brush border membrane sodium-phosphate cotransporter expression as sodium-phosphate cotransporter 2a and 2c expression levels was similar to wild-type levels. Plasma parathyroid hormone and fibroblast growth factor 23 levels were not different; however, fibroblast growth factor 23 mRNA levels were significantly increased in femur homogenates from Uchl1-/- mice. Full-length and soluble α-klotho levels were comparable in kidneys from wild-type and Uchl1-/- mice; however, soluble α-klotho was reduced in Uchl1-/- mice urine. Consistent with unchanged components of 1,25(OH)2D3 metabolism (i.e., CYP27B1 and CYP24A1), sodium-phosphate cotransporter 2b protein levels were not different in ileum brush borders from Uchl1-/- mice, suggesting that the intestine is not the source of hyperphosphatemia. Nonetheless, when Uchl1-/- mice were fed a low-phosphate diet, plasma phosphate, urinary phosphate, and fractional excretion of phosphate were significantly attenuated and comparable to levels of low-phosphate diet-fed wild-type mice. Our findings demonstrate that Uchl1-deleted mice exhibit perturbed phosphate homeostasis, likely consequent to decreased urinary soluble α-klotho, which can be rescued with a low-phosphate diet. Uchl1-/- mice may provide a useful mouse model to study mild perturbations in phosphate homeostasis.

Reversible modulation of SIRT1 activity in a mouse strain.

The SIRT1 protein deacetylase is reported to have a remarkably wide spectrum of biological functions affecting such varied processes as aging, cancer, metabolism, neurodegeneration and immunity. However, the SIRT1 literature is also full of contradictions. To help establish the role(s) of SIRT1 in these and other biological processes, we set out to create a mouse in which the SIRT1 activity could be toggled between on and off states by fusing the estrogen receptor ligand-binding domain (ER) to the C terminus of the SIRT1 protein. We found that the catalytic activity of the SIRT1-ER fusion protein increased 4-5 fold in cells treated with its ligand, 4-hydroxy-tamoxifen (4OHT). The 4OHT-induced activation of SIRT1-ER was due in large part to a 2 to 4-fold increase in abundance of the SIRT1-ER protein in cells in culture and in tissues in vivo. This increase is reversible and is a consequence of 4OHT-induced stabilization of the SIRT1-ER protein. Since changes in SIRT1 level or activity of 2-4 fold are frequently reported to be sufficient to affect its biological functions, this mouse should be helpful in establishing the causal relationships between SIRT1 and the diseases and processes it affects.

Evolution of the highly networked deubiquitinating enzymes USP4, USP15, and USP11.

BACKGROUND: USP4, USP15 and USP11 are paralogous deubiquitinating enzymes as evidenced by structural organization and sequence similarity. Based on known interactions and substrates it would appear that they have partially redundant roles in pathways vital to cell proliferation, development and innate immunity, and elevated expression of all three has been reported in various human malignancies. The nature and order of duplication events that gave rise to these extant genes has not been determined, nor has their functional redundancy been established experimentally at the organismal level. METHODS: We have employed phylogenetic and syntenic reconstruction methods to determine the chronology of the duplication events that generated the three paralogs and have performed genetic crosses to evaluate redundancy in mice. RESULTS: Our analyses indicate that USP4 and USP15 arose from whole genome duplication prior to the emergence of jawed vertebrates. Despite having lower sequence identity USP11 was generated later in vertebrate evolution by small-scale duplication of the USP4-encoding region. While USP11 was subsequently lost in many vertebrate species, all available genomes retain a functional copy of either USP4 or USP15, and through genetic crosses of mice with inactivating mutations we have confirmed that viability is contingent on a functional copy of USP4 or USP15. Loss of ubiquitin-exchange regulation, constitutive skipping of the seventh exon and neural-specific expression patterns are derived states of USP11. Post-translational modification sites differ between USP4, USP15 and USP11 throughout evolution. CONCLUSIONS: In isolation sequence alignments can generate erroneous USP gene phylogenies. Through a combination of methodologies the gene duplication events that gave rise to USP4, USP15, and USP11 have been established. Although it operates in the same molecular pathways as the other USPs, the rapid divergence of the more recently generated USP11 enzyme precludes its functional interchangeability with USP4 and USP15. Given their multiplicity of substrates the emergence (and in some cases subsequent loss) of these USP paralogs would be expected to alter the dynamics of the networks in which they are embedded.

Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.

OBJECTIVES: To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). METHODS: Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). RESULTS: Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. CONCLUSIONS: Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are potential therapeutic targets in OA.

Loss of UCHL1 promotes age-related degenerative changes in the enteric nervous system.

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging.

Ubiquitin C-terminal hydrolase L1 deletion ameliorates glomerular injury in mice with ACTN4-associated focal segmental glomerulosclerosis.

Renal ubiquitin C-terminal hydrolase L1 (UCHL1) is upregulated in a subset of human glomerulopathies, including focal segmental glomerulosclerosis (FSGS), where it may serve to promote ubiquitin pools for degradation of cytotoxic proteins. In the present study, we tested whether UCHL1 is expressed in podocytes of a mouse model of ACTN4-associated FSGS. Podocyte UCHL1 protein was detected in glomeruli of K256E-ACTN4(pod+)/UCHL1+/+ mice. UCHL1+/- mice were intercrossed with K256E-ACTN4(pod+) mice and monitored for features of glomerular disease. 10-week-old K256E-ACTN4(pod+)/UCHL1-/- mice exhibited significantly ameliorated albuminuria, glomerulosclerosis, tubular pathology and blood pressure. Interestingly, while UCHL1 deletion diminished both tubular and glomerular apoptosis, WT1-positive nuclei were unchanged. Finally, UCHL1 levels correlated positively with poly-ubiquitinated proteins but negatively with K256E-α-actinin-4 levels, implying reduced K256E-α-actinin-4 proteolysis in the absence of UCHL1. Our data suggest that UCHL1 upregulation in ACTN4-associated FSGS fuels the proteasome and that UCHL1 deletion may impair proteolysis and thereby preserve K256E/wt-α-actinin-4 heterodimers, maintaining podocyte cytoskeletal integrity and protecting the glomerular filtration barrier.

SirT1 catalytic activity is required for male fertility and metabolic homeostasis in mice.

The protein encoded by the sirt1 gene is an enzyme, SirT1, that couples the hydrolysis of NAD(+) to the deacetylation of acetyl-lysine residues in substrate proteins. Mutations of the sirt1 gene that fail to encode protein have been introduced into the mouse germ line, and the animals homozygous for these null mutations have various physiological abnormalities. To determine which of the characteristics of these sirt1(-/-) mice are a consequence of the absence of the catalytic activity of the SirT1 protein, we created a mouse strain carrying a point mutation (H355Y) that ablates the catalytic activity but does not affect the amount of the SirT1 protein. Mice carrying point mutations in both sirt1 genes, sirt1(Y/Y), have a phenotype that is overlapping but not identical to that of the sirt1-null animals. The sirt1(Y/Y) phenotype is significantly milder than that seen in the sirt1(-/-) animals. For example, female sirt1(Y/Y) animals are fertile, while sirt1(-/-) females are sterile. On the other hand, both sirt1(-/-) and sirt1(Y/Y) male mice are sterile and hypermetabolic. We report that sirt1(Y/Y) mice respond aberrantly to caloric restriction, although the effects are more subtle than seen in sirt1(-/-) mice. Thus, the SirT1 protein has functions that can be attributed to the catalytic activity of the protein, as well as other functions that are conferred by the protein itself.

Identification and characterization of Hedgehog modulator properties after functional coupling of Smoothened to G15.

The seven-transmembrane receptor Smoothened (Smo) transduces the signal initiated by Hedgehog (Hh) morphogen binding to the receptor Patched (Ptc). We have reinvestigated the pharmacological properties of reference molecules acting on the Hh pathway using various Hh responses and a novel functional assay based on the coexpression of Smo with the alpha subunit of the G15 protein in HEK293 cells. The measurement of inositol phosphate (IP) accumulation shows that Smo has constitutive activity, a response blocked by Ptc which indicates a functional Hh receptor complex. Interestingly, the antagonists cyclopamine, Cur61414, and SANT-1 display inverse agonist properties and the agonist SAG has no effect at the Smo-induced IP response, but converts Ptc-mediated inactive forms of Smo into active ones. An oncogenic Smo mutant does not mediate an increase in IP response, presumably reflecting its inability to reach the cell membrane. These studies identify novel properties of molecules displaying potential interest in the treatment of various cancers and brain diseases, and demonstrate that Smo is capable of signaling through G15.

Transfer of differentiation signal by membrane microvesicles harboring hedgehog morphogens.

Hedgehog (Hh) proteins are considered diffusible morphogens that can be membrane anchored, playing an essential role during development. Here we show that Hh morphogens are associated with microvesicles (MVs) shed from the plasma membrane of apoptotic/stimulated T cells. Hh+ MVs induced differentiation of human K562 pluripotent erythroleukemic cells toward megakaryocytic lineage, as testified to by the expression of alpha(IIb)beta3 integrin and CD42b and changes in the cell cycle. Blocking Hh pathway with either cyclopamine, neutralizing antibodies, or inhibitors of the protein kinase A pathway resulted in the inhibition of these effects. Activation of Hh signaling by SAG, a synthetic agonist, mimicked effects of Hh+ MVs on K562 cells. Human Hh+ MVs, circulating in vivo or derived from apoptotic/stimulated lymphocytes from healthy and diabetic individuals, elicited K562 cell differentiation, also inhibited by cyclopamine. In addition, Hh+ MV-treated primary human CD34+ cells presented an increase of CD41+ CD42- and CD41+ CD42+ megakaryocytic populations with an increase of corresponding polyploidy, both being reduced by blockers of the Hh pathway. Because virtually all cell types undergo plasma membrane remodeling when stimulated, derived MVs can therefore be considered true vectors in the transfer of morphogen-borne biologic information to remote responsive cells, and thereby contribute to the maintenance of homeostasis.

In vitro effects of strontium ranelate on the extracellular calcium-sensing receptor.

The extracellular calcium-sensing receptor (CaSR) is activated by divalent cations and might mediate some of the effects of strontium ranelate, a new drug for the prevention and treatment of post-menopausal osteoporosis. Here, we showed that the maximal effect of Sr(2+) was comparable to that observed for Ca(2+) for both the cloned rat CaSR expressed in Chinese hamster ovary [CHO(CaSR)] cells and the mouse CaSR constitutively expressed in AtT-20 cells as measured by the accumulation of [(3)H]inositol phosphates (IP) resulting from CaSR activation. Strontium ranelate also displayed comparable agonist activity for the CaSR in both cell lines. Sodium ranelate did not stimulate the IP response in CHO(CaSR) cells. The IP response resulting from activation of other G-protein-coupled receptors was potentiated by Sr(2+), suggesting that entry of Sr(2+) into the cells might influence phospholipase C activity. Modulation of the CaSR activity in bone cells by strontium ranelate may contribute to its reported antiosteoporotic effects.

Functional analysis of novel sonic hedgehog gene mutations identified in basal cell carcinomas from xeroderma pigmentosum patients.

Altered sonic hedgehog (SHH) signaling is crucial in the development of basal cell carcinomas (BCC), the most common human cancer. Mutations in SHH signal transducers, PATCHED and SMOOTHENED, have already been identified, but SHH mutations are extremely rare; only 1 was detected in 74 sporadic BCCs. We present data showing unique SHH mutations in BCCs from repair-deficient, skin cancer-prone xeroderma pigmentosum (XP) patients, which are characterized by high levels of UV-specific mutations in key genes involved in skin carcinogenesis, including PATCHED and SMOOTHENED. Thus, 6 UV-specific SHH mutations were detected in 5 of 33 XP BCCs. These missense SHH alterations are not activating mutations for its postulated proto-oncogene function, as the mutant SHH proteins do not show transforming activity and induce differentiation or stimulate proliferation to the same level as the wild-type protein. Structural modeling studies of the 4 proteins altered at the surface residues, G57S, G64K, D147N, and R155C, show that they do not effect the protein conformation. Interestingly, they are all located on one face of the compact SHH protein suggesting that they may have altered affinity for different partners, which may be important in altering other functions. Additional functional analysis of the SHH mutations found in vivo in XP BCCs will help shed light on the role of SHH in skin carcinogenesis. In conclusion, we report for the first time, significant levels of SHH mutations found only in XP BCCs and none in squamous cell carcinomas, indicating their importance in the specific development of BCCs.