REDD1 interacts with AIF and regulates mitochondrial reactive oxygen species generation in the keratinocyte response to UVB.

Non-melanoma skin cancer (NMSC) incidence is rising, especially in high-risk, immunocompromised groups such as organ transplant patients, who often develop numerous, aggressive cutaneous squamous cell carcinomas. Identifying the pathways that support NMSC development will result in new approaches for prevention and therapy. Our goal is to define the function of REDD1 (Regulated in DNA Damage and Development 1) in the UVB stress response. REDD1 is rapidly induced by a variety of stressors to repress mechanistic target of rapamycin complex I (mTORC1), and it has been reported that REDD1 loss causes dysfunctional mitochondria with increased reactive oxygen species (ROS) and impaired oxidative phosphorylation (OXPHOS). We now show that knockout of REDD1 in human keratinocytes sensitizes them to UVB-induced apoptosis in an mTORC1-independent manner and intensifies mitochondrial ROS generation. Upon REDD1 knockout, we observe reduced levels of apoptosis inducing factor (AIF), a mitochondrial intermembrane space NADH oxidase that is required for electron transport chain Complex I biogenesis. Further, we show that keratinocyte REDD1 interacts with both AIF and the mitochondrial import protein CHCHD4, a direct binding partner of AIF that ensures functional OXPHOS. Our results support the hypothesis that REDD1 is part of a mitochondrial complex that protects cells from UVB-induced ROS toxicity and suggest novel therapeutic targets for prevention and therapy of NMSC.

mTORC2 confers neuroprotection and potentiates immunity during virus infection.

Herpes simplex virus type-1 (HSV-1) causes ocular and orofacial infections. In rare cases, HSV-1 can cause encephalitis, which leads to permanent brain injuries, memory loss or even death. Host factors protect humans from viral infections by activating the immune response. However, factors that confer neuroprotection during viral encephalitis are poorly understood. Here we show that mammalian target of rapamycin complex 2 (mTORC2) is essential for the survival of experimental animals after ocular HSV-1 infection in vivo. We find the loss of mTORC2 causes systemic HSV-1 infection due to defective innate and adaptive immune responses, and increased ocular and neuronal cell death that turns lethal for the infected mice. Furthermore, we find that mTORC2 mediated cell survival channels through the inactivation of the proapoptotic factor FoxO3a. Our results demonstrate how mTORC2 potentiates host defenses against viral infections and implicate mTORC2 as a necessary factor for survival of the infected host.

Knocking down raptor in human keratinocytes affects ornithine decarboxylase in a post-transcriptional Manner following ultraviolet B exposure.

Non-melanoma skin cancer (NMSC) is the most common form of cancer. Ultraviolet-B (UVB) radiation has been shown to be a complete carcinogen in the development of NMSC. The mammalian target of rapamycin complex 1 (mTORC1) is upregulated by UVB. Ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, is also upregulated in response to UVB. However, the interplay between these two pathways after UVB exposure remains unclear. The studies described here compare mRNA stability between normal human keratinocytes (HaCaT cells) and HaCaT cells with low levels of raptor to investigate whether the induction of ODC by UVB is dependent on mTORC1. We show that the knockdown of mTORC1 activity led to decreased levels of ODC protein both before and after exposure to 20 mJ/cm2 UVB. ODC mRNA was less stable in cells with decreased mTORC1 activity. Polysome profiles revealed that the initiation of ODC mRNA translation did not change in UVB-treated cells. We have shown that the ODC transcript is stabilized by the RNA-binding protein human antigen R (HuR). To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability in human keratinocytes exposed to UVB. We show an increased cytoplasmic localization of HuR after UVB exposure in wild-type cells. The ablation of HuR via CRISPR/Cas9 did not alter the stability of the ODC message, suggesting the involvement of other trans-acting factors. These data suggest that in human keratinocytes, ODC mRNA stability is regulated, in part, by an mTORC1-dependent mechanism after UVB exposure.

Hypermethylation of FOXA1 and allelic loss of PTEN drive squamous differentiation and promote heterogeneity in bladder cancer.

Intratumoral heterogeneity in bladder cancer is a barrier to accurate molecular sub-classification and treatment efficacy. However, individual cellular and mechanistic contributions to tumor heterogeneity are controversial. We examined potential mechanisms of FOXA1 and PTEN inactivation in bladder cancer and their contribution to tumor heterogeneity. These analyses were complemented with inactivation of FOXA1 and PTEN in intermediate and luminal mouse urothelium. We show inactivation and reduced expression of FOXA1 and PTEN is prevalent in human disease, where PTEN and FOXA1 are downregulated by allelic loss and site-specific DNA hypermethylation, respectively. Conditional inactivation of both Foxa1 and Pten in intermediate/luminal cells in mice results in development of bladder cancer exhibiting squamous features as well as enhanced sensitivity to a bladder-specific carcinogen. In addition, FOXA1 is hypermethylated in basal bladder cancer cell lines, and this is reversed by treatment with DNA methyltransferase inhibitors. By integrating human correlative and in vivo studies, we define a critical role for PTEN loss and epigenetic silencing of FOXA1 in heterogeneous human disease and show genetic targeting of luminal/intermediate cells in mice drives squamous differentiation.

Knockout of Raptor destabilizes ornithine decarboxylase mRNA and decreases binding of HuR to the ODC transcript in cells exposed to ultraviolet-B irradiation.

Non-melanoma skin cancer (NMSC) is the most commonly diagnosed cancer in the United States. Ultraviolet-B (UVB) irradiation is the primary carcinogen responsible for stimulating NMSC development. Ornithine Decarboxylase (ODC), the first rate-limiting enzyme in the synthesis of polyamines, is upregulated in response to a variety of proliferation stimuli, including UVB exposure. Our previous studies have demonstrated regulation of ODC synthesis by the mammalian target of rapamycin complex 1 (mTORC1) in cells transformed by oncogenic Ras. The goal of these studies was to better understand the link between mTORC1 and ODC in nontransformed cells treated with UVB. We show that the ablation of mTORC1 activity by conditional knockout of its essential component Raptor led to decreased levels of ODC protein both before and after exposure to 10 mJ/cm2 UVB. Moreover, ODC mRNA was destabilized in the absence of Raptor, suggesting post-transcriptional regulation. We have previously shown that the ODC transcript is stabilized by the RNA binding protein (RBP) human antigen R (HuR), and the intracellular localization of HuR responds to changes in mTORC1 activity. To expand these studies, we investigated whether HuR functions to regulate ODC mRNA stability after UVB exposure. Our results show an increased localization of HuR to the cytoplasm after UVB exposure in wild-type cells compared to Raptor knockout cells, and this is accompanied by greater association of HuR with the ODC transcript. These data suggest that the localization of HuR in response to UVB is influenced, at least in part, by mTORC1 and that HuR can bind to and stabilize ODC mRNA after UVB exposure in an mTORC1-dependent manner.

Inhibition of mTORC2 enhances UVB-induced apoptosis in keratinocytes through a mechanism dependent on the FOXO3a transcriptional target NOXA but independent of TRAIL.

The primary cause of non-melanoma skin cancer (NMSC) is ultraviolet B (UVB) radiation. We have shown previously that mTORC2 inhibition sensitizes keratinocytes to UVB-induced apoptosis mediated by the transcription factor FOXO3a. FOXO3a is a key regulator of apoptosis and a tumor suppressor in several cancer types. Activation of FOXO3a promotes apoptosis through the coordinated expression of a variety of target genes, including TRAIL and NOXA. We hypothesized that in the setting of mTORC2 inhibition, the UVB-induced expression of these factors would lead to apoptosis in a FOXO3a-dependent manner. Using spontaneously immortalized human keratinocytes (HaCaT cells), we observed that both TRAIL and NOXA expression increased in cells exposed to UVB and the TOR kinase inhibitor Torin 2. Similar to knockdown of FOXO3a, NOXA knockdown reversed the sensitization to UVB-induced apoptosis caused by mTORC2 inhibition. In contrast, loss of TRAIL by either knockdown or knockout actually enhanced expression of nuclear FOXO3a, which maintained apoptosis. These surprising results are not due to faulty death receptor signaling in HaCaT cells, as we found that the cells undergo extrinsic apoptosis in response to treatment with recombinant TRAIL. Even more striking, TRAIL knockout cells were sensitized to recombinant TRAIL-induced apoptosis compared to wild-type HaCaT cells, with the largest increase occurring in the presence of mTORC2 inhibition. Taken together, these studies provide strong evidence that mTORC2 controls UVB-induced apoptosis by regulating NOXA expression downstream of FOXO3a. Moreover, FOXO3a transcriptional activation by mTORC2 inhibitors may be a valuable target for prevention or therapy of NMSC, especially in cases with low endogenous TRAIL.

The ODC 3'-Untranslated Region and 5'-Untranslated Region Contain cis-Regulatory Elements: Implications for Carcinogenesis.

It has been hypothesized that both the 3'-untranslated region (3'UTR) and the 5'-untranslated region (5'UTR) of the ornithine decarboxylase (ODC) mRNA influence the expression of the ODC protein. Here, we use luciferase expression constructs to examine the influence of both UTRs in keratinocyte derived cell lines. The ODC 5'UTR or 3'UTR was cloned into the pGL3 control vector upstream or downstream of the luciferase reporter gene, respectively, and luciferase activity was measured in both non-tumorigenic and tumorigenic mouse keratinocyte cell lines. Further analysis of the influence of the 3'UTR on luciferase activity was accomplished through site-directed mutagenesis and distal deletion analysis within this region. Insertion of either the 5'UTR or 3'UTR into a luciferase vector resulted in a decrease in luciferase activity when compared to the control vector. Deletion analysis of the 3'UTR revealed a region between bases 1969 and 2141 that was inhibitory, and mutating residues within that region increased luciferase activity. These data suggest that both the 5'UTR and 3'UTR of ODC contain cis-acting regulatory elements that control intracellular ODC protein levels.

Molecular signaling cascades involved in nonmelanoma skin carcinogenesis.

Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide and the incidence continues to rise, in part due to increasing numbers in high-risk groups such as organ transplant recipients and those taking photosensitizing medications. The most significant risk factor for NMSC is ultraviolet radiation (UVR) from sunlight, specifically UVB, which is the leading cause of DNA damage, photoaging, and malignant transformation in the skin. Activation of apoptosis following UVR exposure allows the elimination of irreversibly damaged cells that may harbor oncogenic mutations. However, UVR also activates signaling cascades that promote the survival of these potentially cancerous cells, resulting in tumor initiation. Thus, the UVR-induced stress response in the skin is multifaceted and requires coordinated activation of numerous pathways controlling DNA damage repair, inflammation, and kinase-mediated signal transduction that lead to either cell survival or cell death. This review focuses on the central signaling mechanisms that respond to UVR and the subsequent cellular changes. Given the prevalence of NMSC and the resulting health care burden, many of these pathways provide promising targets for continued study aimed at both chemoprevention and chemotherapy.

Destabilization of the ornithine decarboxylase mRNA transcript by the RNA-binding protein tristetraprolin.

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. In a normal physiological state, ODC is tightly regulated. However, during neoplastic transformation, ODC expression becomes upregulated. The studies described here show that the ODC mRNA transcript is destabilized by the RNA-binding protein tristetraprolin (TTP). We show that TTP is able to bind to the ODC mRNA transcript in both non-transformed RIE-1 cells and transformed Ras12V cells. Moreover, using mouse embryonic fibroblast cell lines that are devoid of a functional TTP protein, we demonstrate that in the absence of TTP both ODC mRNA stability and ODC enzyme activity increase when compared to wild-type cells. Finally, we show that the ODC 3' untranslated region contains cis acting destabilizing elements that are affected by, but not solely dependent on, TTP expression. Together, these data support the hypothesis that TTP plays a role in the post-transcriptional regulation of the ODC mRNA transcript.

Negative regulation of the FOXO3a transcription factor by mTORC2 induces a pro-survival response following exposure to ultraviolet-B irradiation.

Exposure to ultraviolet-B (UVB) irradiation, the principal cause of non-melanoma skin cancer (NMSC), activates both the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2. We have previously reported that UVB-induced keratinocyte survival is dependent on mTORC2, though the specific mechanism is not well understood. FOXO3a is an important transcription factor involved in regulating cell survival. The activity of FOXO3a is reduced as a result of protein kinase B (AKT/PKB) activation, which is downstream of mTORC2; however, the specific function of FOXO3a during UVB-induced apoptosis is unclear. In this study, we establish that in cells with wild-type mTORC2 activity, FOXO3a is quickly phosphorylated in response to UVB and sequestered in the cytoplasm. In contrast, loss of mTORC2 causes FOXO3a to be localized to the nucleus and sensitizes cells to UVB-induced apoptosis. Furthermore, this sensitization is rescued by knockdown of FOXO3a. Taken together, these studies provide strong evidence that inhibition of mTORC2 enhances UVB-induced apoptosis in a FOXO3a-dependent manner, and suggest that FOXO3a activation by mTORC2 inhibitors may be a valuable chemopreventive target in NMSC.

Skin Carcinogenesis Studies Using Mouse Models with Altered Polyamines.

Nonmelanoma skin cancer (NMSC) is a major health concern worldwide. With increasing numbers in high-risk groups such as organ transplant recipients and patients taking photosensitizing medications, the incidence of NMSC continues to rise. Mouse models of NMSC allow us to better understand the molecular signaling cascades involved in skin tumor development in order to identify novel therapeutic strategies. Here we review the models designed to determine the role of the polyamines in NMSC development and maintenance. Elevated polyamines are absolutely required for tumor growth, and dysregulation of their biosynthetic and catabolic enzymes has been observed in NMSC. Studies using mice with genetic alterations in epidermal polyamines suggest that they play key roles in tumor promotion and epithelial cell survival pathways, and recent clinical trials indicate that pharmacological inhibitors of polyamine metabolism show promise in individuals at high risk for NMSC.

Conditional disruption of rictor demonstrates a direct requirement for mTORC2 in skin tumor development and continued growth of established tumors.

Activation of signaling dependent on the mammalian target of rapamycin (mTOR) has been demonstrated in a variety of human malignancies, and our previous work suggests that mTOR complex (mTORC) 1 and mTORC2 may play unique roles in skin tumorigenesis. The purpose of these studies was to investigate the function of mTORC2-dependent pathways in skin tumor development and the maintenance of established tumors. Using mice that allow spatial and temporal control of mTORC2 in epidermis by conditional knockout of its essential component Rictor, we studied the effect of mTORC2 loss on both epidermal proliferation and chemical carcinogenesis. The results demonstrate that mTORC2 is dispensable for both normal epidermal proliferation and the hyperproliferative response to treatment with tetradecanoyl phorbol acetate (TPA). In contrast, deletion of epidermal Rictor prior to initiation in DMBA/TPA chemical carcinogenesis was sufficient to dramatically delay tumor development and resulted in reduced tumor number and size compared with control groups. Silencing of Rictor expression in tumor-bearing animals triggered regression of established tumors and increased caspase-3 cleavage without changes in proliferation. In vitro experiments demonstrate an increased sensitivity to caspase-dependent apoptosis in the absence of rictor, which is dependent on mTORC2 signaling. These studies demonstrate that mTORC2 activation is essential for keratinocyte survival, and suggest that inhibition of mTORC2 has value in chemoprevention by eliminating carcinogen-damaged cells during the early stages of tumorigenesis, and in therapy of existing tumors by restricting critical pro-survival pathways.

Inhibition of mTOR suppresses UVB-induced keratinocyte proliferation and survival.

UV radiation is the major risk factor for developing skin cancer, the most prevalent cancer worldwide. Several studies indicate that mTOR signaling is activated by UVB and may play an important role in skin tumorigenesis. mTOR exists in two functionally and compositionally distinct protein complexes: the rapamycin-sensitive mTOR complex 1 (mTORC1) and the rapamycin-resistant mTOR complex 2 (mTORC2). The purpose of these studies was to investigate the roles of the two mTOR complexes in UVB-mediated proliferation and apoptosis in the skin. We used rapamycin, a pharmacologic inhibitor of mTORC1, and an inducible mTOR-deficient (K5-CreER(T2);mTOR(fl/fl)) mouse model that allows epidermal-specific disruption of mTOR following topical treatment with 4-hydroxytamoxifen (4OHT). Rapamycin blocked UVB-induced phosphorylation of S6K, the downstream target of mTORC1, and significantly reduced UVB-stimulated epidermal proliferation and cell-cycle progression, but had no effect on cell death. In contrast, mTOR deletion, which attenuated UVB-induced phosphorylation of both S6K and the mTORC2 target AKT(Ser473), significantly increased apoptosis both in vivo and in keratinocyte cultures, in addition to reducing hyperproliferation following UVB irradiation. The role of mTORC2 in UVB-induced prosurvival signaling was verified in Rictor(-/-) mouse embryo fibroblasts, which lack functional mTORC2 and were more sensitive to UVB-induced apoptosis than controls. These studies show that mTORC1 and mTORC2 play unique but complementary roles in controlling proliferation and apoptosis in the skin. Our findings underscore the importance of both mTOR complexes in mediating UVB-induced signaling in keratinocytes and provide new insight into the pathogenesis of skin cancer.

S-adenosylmethionine decarboxylase overexpression inhibits mouse skin tumor promotion.

Neoplastic growth is associated with increased polyamine biosynthetic activity and content. Tumor promoter treatment induces the rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (AdoMetDC), and targeted ODC overexpression is sufficient for tumor promotion in initiated mouse skin. We generated a mouse model with doxycycline (Dox)-regulated AdoMetDC expression to determine the impact of this second rate-limiting enzyme on epithelial carcinogenesis. TetO-AdoMetDC (TAMD) transgenic founders were crossed with transgenic mice (K5-tTA) that express the tetracycline-regulated transcriptional activator within basal keratinocytes of the skin. Transgene expression in TAMD/K5-tTA mice was restricted to keratin 5 (K5) target tissues and silenced upon Dox treatment. AdoMetDC activity and its product, decarboxylated AdoMet, both increased approximately 8-fold in the skin. This enabled a redistribution of the polyamines that led to reduced putrescine, increased spermine, and an elevated spermine:spermidine ratio. Given the positive association between polyamine biosynthetic capacity and neoplastic growth, it was somewhat surprising to find that TAMD/K5-tTA mice developed significantly fewer tumors than controls in response to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate chemical carcinogenesis. Importantly, tumor counts in TAMD/K5-tTA mice rebounded to nearly equal the levels in the control group upon Dox-mediated transgene silencing at a late stage of tumor promotion, which indicates that latent viable initiated cells remain in AdoMetDC-expressing skin. These results underscore the complexity of polyamine modulation of tumor development and emphasize the critical role of putrescine in tumor promotion. AdoMetDC-expressing mice will enable more refined spatial and temporal manipulation of polyamine biosynthesis during tumorigenesis and in other models of human disease.

Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy.

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to ß-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to ß-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Ornithine decarboxylase mRNA is stabilized in an mTORC1-dependent manner in Ras-transformed cells.

Upon Ras activation, ODC (ornithine decarboxylase) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumours. In the present study we compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed RIE-1 (rat intestinal epithelial) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC mRNA was stabilized 8-fold. Treatment with the specific mTORC1 [mTOR (mammalian target of rapamycin) complex 1] inhibitor rapamycin or siRNA (small interfering RNA) knockdown of mTOR destabilized the ODC mRNA, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA-binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3'UTR (untranslated region) results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3'UTR that may act as regulatory sequences. Analysis of ODC 3'UTR deletion constructs suggests that cis-acting elements between base 1969 and base 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent tumour development.

Posttranscriptional regulation of ornithine decarboxylase.

Activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and intracellular levels of ODC protein are controlled very tightly. Numerous studies have described ODC regulation at the levels of transcription, translation, and protein degradation in normal cells and dysregulation of these processes in response to oncogenic stimuli. Although posttranscriptional regulation of ODC has been well documented, the RNA binding proteins (RBPs) that interact with ODC mRNA and control synthesis of the ODC protein have not been defined. Using Ras-transformed rat intestinal epithelial cells (Ras12V cells) as a model, we have begun identifying the RBPs that associate with the ODC transcript. Binding of RBPs could potentially regulate ODC synthesis by either changing mRNA stability or rate of mRNA translation. Techniques for measuring RBP binding and translation initiation are described here. Targeting control of ODC translation or mRNA decay could be a valuable method of limiting polyamine accumulation and subsequent tumor development in a variety of cancers.

Cytoplasmic accumulation of the RNA-binding protein HuR stabilizes the ornithine decarboxylase transcript in a murine nonmelanoma skin cancer model.

Ornithine decarboxylase (ODC) is the first and usually rate-limiting enzyme in the polyamine biosynthetic pathway. Under normal physiological conditions, polyamine content and ODC enzyme activity are highly regulated. However, the induction of ODC activity is an early step in neoplastic transformation. The studies described here use normal mouse keratinocytes (C5N cells), and spindle carcinoma cells (A5 cells) to explore the regulation of ODC in nonmelanoma skin cancer development. Previous results have shown that induction of ODC activity is both necessary and sufficient for the promotion of skin tumors. We see a marked increase in ODC enzyme activity in A5 cells compared with C5N keratinocytes, which correlates with a 4-fold stabilization of ODC mRNA. These data suggest that ODC is post-transcriptionally regulated in skin tumor development. Thus, we sought to investigate whether the ODC transcript interacts with the RNA-binding protein HuR, which is known to bind to and stabilize its target mRNAs. We show that HuR is able to bind to the ODC 3'-UTR in A5 cells but not in C5N cells. Immunofluorescence results reveal that HuR is present in both the nucleus and cytoplasm of A5 cells, whereas C5N cells exhibit strictly nuclear localization of HuR. Knockdown experiments in A5 cells showed that when HuR is depleted, ODC RNA becomes less stable, and ODC enzyme activity decreases. Together, these data support the hypothesis that HuR plays a causative role in ODC up-regulation during nonmelanoma skin cancer development by binding to and stabilizing the ODC transcript.

Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts.

Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells.

Polyamine homeostasis in arginase knockout mice.

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N(1)-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues.