CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3β/β-catenin-WIP1 pathway

Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2^an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2^+/+ and Cdk5rap2^+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.Subject terms: Senescence, Diseases     

p38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21^Cip1. However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21^Cip1 promoter activity and p21^Cip1 mRNA/protein expression. We further show that exogenously expressed p38δ increases p21^Cip1 mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21^Cip1 levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21^Cip1 gene expression.     

Genetic disruption of Abl nuclear import reduces renal apoptosis in a mouse model of cisplatin-induced nephrotoxicity

DNA damage activates nuclear Abl tyrosine kinase to stimulate intrinsic apoptosis in cancer cell lines and mouse embryonic stem cells. To examine the in vivo function of nuclear Abl in apoptosis, we generated Abl-μNLS (μ, mutated in nuclear localization signals) mice. We show here that cisplatin-induced apoptosis is defective in the renal proximal tubule cells (RPTC) from the Abl^μ/μ mice. When injected with cisplatin, we found similar levels of platinum in the Abl^+/+ and the Abl^μ/μ kidneys, as well as similar initial inductions of p53 and PUMAα expression. However, the accumulation of p53 and PUMAα could not be sustained in the Abl^μ/μ kidneys, leading to reductions in renal apoptosis and tubule damage. Co-treatment of cisplatin with the Abl kinase inhibitor, imatinib, reduced the accumulation of p53 and PUMAα in the Abl^+/+ but not in the Abl^μ/μ kidneys. The residual apoptosis in the Abl^μ/μ mice was not further reduced in the Abl^μ/μ; p53^−/− double-mutant mice, suggesting that nuclear Abl and p53 are epistatic to each other in this apoptosis response. Although apoptosis and tubule damage were reduced, cisplatin-induced increases in phospho-Stat-1 and blood urea nitrogen were similar between the Abl^+/+ and the Abl^μ/μ kidneys, indicating that RPTC apoptosis is not the only factor in cisplatin-induced nephrotoxicity. These results provide in vivo evidence for the pro-apoptotic function of Abl, and show that its nuclear localization and tyrosine kinase activity are both required for the sustained expression of p53 and PUMAα in cisplatin-induced renal apoptosis.     

The Tumor Suppressor Gene,RASSF1A,Is Essential for Protection against Inflammation -Induced Injury

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a^+/−, Rassf1a^−/− and an intestinal epithelial cell specific knockout mouse (Rassf1a ^IEC-KO) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a^−/− mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a^−/− background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a^−/− mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.     

Rb-dependent cellular senescence,multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK), such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16^Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ.Key words: SSeCKS/Akap12, PKC, senescence, MEF, Rb, Lats1/Warts, p16^Ink4a, Id1, polyploidy, binucleation     

Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland

Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-A^S390A mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis.     

Lipocalin 2 Regulates Brown Fat Activation via a Nonadrenergic Activation Mechanism

In this study, we report that lipocalin 2 (Lcn2), a recently characterized adipokine/cytokine, is a novel regulator of brown adipose tissue (BAT) activation by modulating the adrenergic independent p38 MAPK-PGC-1α-UCP1 pathway. Global Lcn2 knock-out (Lcn2^−/−) mice have defective BAT thermogenic activation caused by cold stimulation and decreased BAT activity under high fat diet-induced obesity. Nevertheless, Lcn2^−/− mice maintain normal sympathetic nervous system activation as evidenced by normal catecholamine release and lipolytic activity in response to cold stimulation. Further studies showed that Lcn2 deficiency impairs peroxisomal and mitochondrial oxidation of lipids and attenuates cold-induced Pgc1a and Ucp1 expression and p38 MAPK phosphorylation in BAT. Moreover, in vitro studies showed that Lcn2 deficiency reduces the thermogenic activity of brown adipocytes. Lcn2^−/− differentiated brown adipocytes have significantly decreased expression levels of brown fat markers, decreased p38 MAPK phosphorylation, and decreased mitochondrial oxidation capacity. However, Lcn2^−/− brown adipocytes have normal norepinephrine-stimulated p38 MAPK and hormone-sensitive lipase phosphorylation and Pgc1a and Ucp1 expression, suggesting an intact β-adrenergic signaling activation. More intriguingly, recombinant Lcn2 was able to significantly stimulate p38 MAPK phosphorylation in brown adipocytes. Activating peroxisome proliferator-activated receptor γ, a downstream effector of PGC-1α, by thiazolidinedione administration fully reverses the BAT function of Lcn2^−/− mice. Our findings provide evidence for the novel role Lcn2 plays in oxidative metabolism and BAT activation via an adrenergic independent mechanism.     

Glycerol-3-phosphate Acyltransferase Isoform-4 (GPAT4) Limits Oxidation of Exogenous Fatty Acids in Brown Adipocytes

Glycerol-3-phosphate acyltransferase-4 (GPAT4) null pups grew poorly during the suckling period and, as adults, were protected from high fat diet-induced obesity. To determine why Gpat4^−/− mice failed to gain weight during these two periods of high fat feeding, we examined energy metabolism. Compared with controls, the metabolic rate of Gpat4^−/− mice fed a 45% fat diet was 12% higher. Core body temperature was 1 ºC higher after high fat feeding. Food intake, fat absorption, and activity were similar in both genotypes. Impaired weight gain in Gpat4^−/− mice did not result from increased heat loss, because both cold tolerance and response to a β3-adrenergic agonist were similar in both genotypes. Because GPAT4 comprises 65% of the total GPAT activity in brown adipose tissue (BAT), we characterized BAT function. A 45% fat diet increased the Gpat4^−/− BAT expression of peroxisome proliferator-activated receptor α (PPAR) target genes, Cpt1α, Pgc1α, and Ucp1, and BAT mitochondria oxidized oleate and pyruvate at higher rates than controls, suggesting that fatty acid signaling and flux through the TCA cycle were enhanced. To assess the role of GPAT4 directly, neonatal BAT preadipocytes were differentiated to adipocytes. Compared with controls, Gpat4^−/− brown adipocytes incorporated 33% less fatty acid into triacylglycerol and 46% more into the pathway of β-oxidation. The increased oxidation rate was due solely to an increase in the oxidation of exogenous fatty acids. These data suggest that in the absence of cold exposure, GPAT4 limits excessive fatty acid oxidation and the detrimental induction of a hypermetabolic state.     

Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21^Cip1 expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21^Cip1 expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21^Cip1 promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21^Cip1 gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21^Cip1 promoter, and that this is associated with enhanced p21^Cip1 expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation.     

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92–495 days) which was much greater than mΔpro/- mice (range 120–250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78–150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.The p53 tumor suppressor is most important for preventing cancers. p53 controls cell fate in response to stress by inducing apoptosis, cell cycle arrest/senescence, DNA repair (reviewed in Braithwaite et al.,^{1, 2} Oren,³ and Speidel⁴) or possibly restricting supply of basic substrates for metabolism.^{5, 6, 7} The regulation of p53 function has recently become more complex with the discovery of 13 isoforms, which may interfere with the normal functioning of full-length (FL) p53.^{8, 9, 10, 11, 12, 13, 14} An alternative promoter in intron 4 generates the Δ133p53 isoforms (Δ133p53α, and with additional alternative splicing in intron 9, Δ133p53β, and Δ133p53γ¹¹).The Δ133p53α isoform is expressed in many tissues, but elevated levels have been found in several cancers.^{11, 15, 16} Although the function(s) of Δ133p53α are not fully understood, growing evidence suggests it may have tumor-promoting capacities. Reducing Δ133p53α levels in the U87MG glioblastoma cell line reduced its ability to migrate and stimulate angiogenesis.¹⁷ Δ133p53α may also interfere with the tumor-suppressor functions of FLp53. The zebrafish ortholog of Δ133p53α, Δ113p53, inhibited p53-mediated apoptosis,¹⁸ and overexpression of Δ133p53α inhibited p53-directed G₁ cell cycle arrest.¹⁶Previously, we reported the construction and characterization of a mouse expressing an N-terminal truncation mutant of p53 (designated Δ122p53) that is very similar to Δ133p53α, providing the first mouse model of the Δ133p53α isoform.^{19, 20} Δ122p53 was found to increase cell proliferation and in p53 null cells transduced with a Δ122p53 expressing retrovirus, inhibited the transactivation of CDKN1a (encoding) p21^CIP1 and MDM2 by FLp53.^{19, 20} As well as elevating cell proliferation, homozygote Δ122p53 mice exhibited a profound pro-inflammatory phenotype, including increased serum interleukin-6 (IL-6) and γ-interferon (γ-IFN), and features of autoimmune disease.^{19, 20} The mice were tumor-prone displaying a complex tumor spectrum, but predominantly B-cell lymphomas and osteosarcomas. Thus, most evidence supports a role for the Δ133p53α isoform as a dominant oncogene that may interfere with normal FLp53 tumor-suppressor functions, but also has additional ''gain-of-function'' properties to promote tumor progression, probably through inflammatory mechanisms.²¹Given the above data, we reasoned that in an environment where p53 tumor-suppression capacity is compromised, such as in the context of the R72P allele^{22, 23, 24} or where p53 levels are reduced,^{25, 26, 27} the influence of Δ133p53α isoform on FLp53 function would be greater, leading to rapid tumor formation with a phenotype that would resemble that of the isoform alone. To test this, we generated mice heterozygous for Δ122p53 and a p53 mutant (mΔpro) that we previously described, that has attenuated tumor-suppressor activity.^{28, 29} The mΔpro mouse model is missing part of the p53 proline rich domain (PRD, amino acids 58–88). These mice are defective for DNA damage-induced apoptosis, and show a delayed and impaired cell cycle arrest response. Homozygous mΔpro mice develop late onset follicular B-cell tumors, while mΔpro heterozygotes developed few tumors in the presence of a wild-type p53 allele, or an early onset T-cell lymphoma in a p53-null background. In the latter case, the onset and tumor spectrum are indistinguishable from p53-null mice.²⁸In the current study, we found that, in contrast to our hypothesis, many Δ122p53/mΔpro mice showed extended survival compared with Δ122p53 homozygotes. In vitro analyses to explain this phenomenon suggested that Δ122p53 allele can enhance mΔpro tumor-suppressor functions, in particular cell cycle arrest.     

Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging

The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63⁺, but not ΔNp63⁺, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal⁺ and p21⁺ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63⁺ TECs, but an increase in ΔNp63⁺ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63⁺ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.     

Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel–Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.