Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion.

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhd(flox/flox) mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma.

Rho-associated coiled-coil-containing kinase 2 deficiency in bone marrow-derived cells leads to increased cholesterol efflux and decreased atherosclerosis.

BACKGROUND: Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil-containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined. METHODS AND RESULTS: The bone marrows from wild-type, ROCK2(+/-), and ROCK2(-/-) mice were transplanted into irradiated recipient low-density lipoprotein receptor(-/-) mice, and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared with wild-type bone marrow-transplanted mice, ROCK2(+/-) bone marrow-transplanted and ROCK2(-/-) bone marrow-transplanted mice showed substantially less lipid accumulation in the aorta (8.46±1.42% and 9.80±2.34% versus 15.64±1.89%; P<0.01 for both) and decreased atherosclerotic lesions in the subaortic sinus (158.1±44.4 and 330.1±109.5×10(3)µm(2) versus 520.2±125.7×10(3)µm(2); P<0.01 for both). These findings correlated with decreased foam cell formation (2.27±0.57 versus 4.10±0.3; P<0.01) and increased cholesterol efflux (17.65±0.6 versus 9.75±0.8; P<0.05) in ROCK2-deficient mice that are mediated, in part, through the peroxisome proliferator-activated receptor-γ/liver X receptor/ATP-binding cassette transporter A1 pathway in macrophages. CONCLUSIONS: ROCK2 contributes to atherosclerosis, in part, by inhibiting peroxisome proliferator-activated receptor-γ-mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.

Histone H4K20/H3K9 demethylase PHF8 regulates zebrafish brain and craniofacial development.

X-linked mental retardation (XLMR) is a complex human disease that causes intellectual disability. Causal mutations have been found in approximately 90 X-linked genes; however, molecular and biological functions of many of these genetically defined XLMR genes remain unknown. PHF8 (PHD (plant homeo domain) finger protein 8) is a JmjC domain-containing protein and its mutations have been found in patients with XLMR and craniofacial deformities. Here we provide multiple lines of evidence establishing PHF8 as the first mono-methyl histone H4 lysine 20 (H4K20me1) demethylase, with additional activities towards histone H3K9me1 and me2. PHF8 is located around the transcription start sites (TSS) of approximately 7,000 RefSeq genes and in gene bodies and intergenic regions (non-TSS). PHF8 depletion resulted in upregulation of H4K20me1 and H3K9me1 at the TSS and H3K9me2 in the non-TSS sites, respectively, demonstrating differential substrate specificities at different target locations. PHF8 positively regulates gene expression, which is dependent on its H3K4me3-binding PHD and catalytic domains. Importantly, patient mutations significantly compromised PHF8 catalytic function. PHF8 regulates cell survival in the zebrafish brain and jaw development, thus providing a potentially relevant biological context for understanding the clinical symptoms associated with PHF8 patients. Lastly, genetic and molecular evidence supports a model whereby PHF8 regulates zebrafish neuronal cell survival and jaw development in part by directly regulating the expression of the homeodomain transcription factor MSX1/MSXB, which functions downstream of multiple signalling and developmental pathways. Our findings indicate that an imbalance of histone methylation dynamics has a critical role in XLMR.

GAMT, a p53-inducible modulator of apoptosis, is critical for the adaptive response to nutrient stress.

The p53 tumor suppressor protein has a well-established role in cell-fate decision-making processes. However, recent discoveries indicate that p53 has a non-tumor-suppressive role. Here we identify guanidinoacetate methyltransferase (GAMT), an enzyme involved in creatine synthesis, as a p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53-dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53-->GAMT upregulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP-generating energy source. These results highlight that p53-dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. The p53-->GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism.

Identification of ROCK1 as an upstream activator of the JIP-3 to JNK signaling axis in response to UVB damage.

Although apoptosis triggered by ultraviolet B (UVB)-mediated activation of the c-Jun N-terminal kinase (JNK) pathway is mediated by both intrinsic and extrinsic pathways, the mechanism of initiation of JNK activation remains obscure. Here, we report the characterization of the JNK-interacting protein 3 (JIP-3) scaffolding protein as an interacting partner of Rho-associated kinase 1 (ROCK1), as determined by tandem affinity protein purification. Upon UVB-induced stress in keratinocytes, ROCK1 was activated, bound to JIP-3, and activated the JNK pathway. Moreover, phosphorylation of JIP-3 by ROCK1 was crucial for the recruitment of JNK. Inhibition of the activity of ROCK1 in keratinocytes resulted in decreased activation of the JNK pathway and thus a reduction in apoptosis. ROCK1(+/-) mice exhibited decreased UVB-mediated activation of JNK and apoptosis relative to wild-type mice. Our findings present a new molecular mechanism by which ROCK1 functions as a UVB sensor that regulates apoptosis, an important event in the prevention of skin cancer.

Prolyl 4-hydroxylation regulates Argonaute 2 stability.

Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the alpha-(P4H-alpha(I)) and beta-(P4H-beta) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-alpha(I) or P4H-beta by short hairpin RNA and P4H-alpha(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.

Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.

Glucose flux through the hexosamine biosynthetic pathway leads to the post-translational modification of cytoplasmic and nuclear proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc). This tandem system serves as a nutrient sensor to couple systemic metabolic status to cellular regulation of signal transduction, transcription, and protein degradation. Here we show that O-GlcNAc transferase (OGT) harbours a previously unrecognized type of phosphoinositide-binding domain. After induction with insulin, phosphatidylinositol 3,4,5-trisphosphate recruits OGT from the nucleus to the plasma membrane, where the enzyme catalyses dynamic modification of the insulin signalling pathway by O-GlcNAc. This results in the alteration in phosphorylation of key signalling molecules and the attenuation of insulin signal transduction. Hepatic overexpression of OGT impairs the expression of insulin-responsive genes and causes insulin resistance and dyslipidaemia. These findings identify a molecular mechanism by which nutritional cues regulate insulin signalling through O-GlcNAc, and underscore the contribution of this modification to the aetiology of insulin resistance and type 2 diabetes.

CDIP, a novel pro-apoptotic gene, regulates TNFalpha-mediated apoptosis in a p53-dependent manner.

We have identified a novel pro-apoptotic p53 target gene named CDIP (Cell Death Involved p53-target). Inhibition of CDIP abrogates p53-mediated apoptotic responses, demonstrating that CDIP is an important p53 apoptotic effector. CDIP itself potently induces apoptosis that is associated with caspase-8 cleavage, implicating the extrinsic cell death pathway in apoptosis mediated by CDIP. siRNA-directed knockdown of caspase-8 results in a severe impairment of CDIP-dependent cell death. In investigating the potential involvement of extrinsic cell death pathway in CDIP-mediated apoptosis, we found that TNF-alpha expression tightly correlates with CDIP expression, and that inhibition of TNF-alpha signaling attenuates CDIP-dependent apoptosis. We also demonstrate that TNF-alpha is upregulated in response to p53 and p53 inducing genotoxic stress, in a CDIP-dependent manner. Consistently, knockdown of TNF-alpha impairs p53-mediated stress-induced apoptosis. Together, these findings support a novel p53 --> CDIP --> TNF-alpha apoptotic pathway that directs apoptosis after exposure of cells to genotoxic stress. Thus, CDIP provides a new link between p53-mediated intrinsic and death receptor-mediated extrinsic apoptotic signaling, providing a novel target for cancer therapeutics aimed at maximizing the p53 apoptotic response of cancer cells to drug therapy.

The protective role of a small GTPase RhoE against UVB-induced DNA damage in keratinocytes.

RhoE, a p53 target gene, was identified as a critical factor for the survival of human keratinocytes in response to UVB. The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton, acting as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations. Unlike typical Rho family proteins, RhoE (also known as Rnd3) is GTPase-deficient and thus expected to be constitutively active. In this study, we investigated the response of cultured human keratinocyte cells to UVB irradiation. RhoE protein levels increase upon exposure to UVB, and ablation of RhoE induction through small interfering RNA resulted in a significant increase in apoptosis and a reduction in the levels of the pro-survival targets p21, Cox-2, and cyclin D1, as well as an increase of reactive oxygen species levels when compared with control cells. These data indicate that RhoE is a pro-survival factor acting upstream of p38, JNK, p21, and cyclin D1. HaCat cells expressing small interfering RNA to p53 indicate that RhoE functions independently of its known associates, p53 and Rho-associated kinase I (ROCK I). Targeted expression of RhoE in epidermis using skin-specific transgenic mouse model resulted in a significant reduction in the number of apoptotic cells following UVB irradiation. Thus, RhoE induction counteracts UVB-induced apoptosis and may serve as a novel target for the prevention of UVB-induced photodamage regardless of p53 status.

RhoE is a pro-survival p53 target gene that inhibits ROCK I-mediated apoptosis in response to genotoxic stress.

The Rho family of GTPases regulates many aspects of cellular behavior through alterations to the actin cytoskeleton . The majority of the Rho family proteins function as molecular switches cycling between the active, GTP-bound and the inactive, GDP-bound conformations . Unlike typical Rho-family proteins, the Rnd subfamily members, including Rnd1, Rnd2, RhoE (also known as Rnd3), and RhoH, are GTPase deficient and are thus expected to be constitutively active . Here, we identify an unexpected role for RhoE/Rnd3 in the regulation of the p53-mediated stress response. We show that RhoE is a transcriptional p53 target gene and that genotoxic stress triggers actin depolymerization, resulting in actin-stress-fiber disassembly through p53-dependent RhoE induction. Silencing of RhoE induction in response to genotoxic stress maintains stress fiber formation and strikingly increases apoptosis, implying an antagonistic role for RhoE in p53-dependent apoptosis. We found that RhoE inhibits ROCK I (Rho-associated kinase I) activity during genotoxic stress and thereby suppresses apoptosis. We demonstrate that the p53-mediated induction of RhoE in response to DNA damage favors cell survival partly through inhibition of ROCK I-mediated apoptosis. Thus, RhoE is anticipated to function by regulating ROCK I signaling to control the balance between cell survival and cell death in response to genotoxic stress.

Discoidin domain receptor 1 receptor tyrosine kinase induces cyclooxygenase-2 and promotes chemoresistance through nuclear factor-kappaB pathway activation.

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by various types of collagens and is known to play a role in cell attachment, migration, survival, and proliferation. However, little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. We report here that DDR1 induces cyclooxygenase-2 (Cox-2) expression resulting in enhanced chemoresistance. Depletion of DDR1-mediated Cox-2 induction using short hairpin RNA (shRNA) results in increased chemosensitivity. We also show that DDR1 activates the nuclear factor-kappaB (NF-kappaB) pathway and blocking this activation by an I kappaB superrepressor mutant results in the ablation of DDR1-induced Cox-2, leading to enhanced chemosensitivity, indicating that DDR1-mediated Cox-2 induction is NF-kappaB dependent. We identify the upstream activating kinases of the NF-kappaB pathway, IKK beta and IKK gamma, as essential for DDR1-mediated NF-kappaB activation, whereas IKK alpha seems to be dispensable. Finally, shRNA-mediated inhibition of DDR1 expression significantly enhanced chemosensitivity to genotoxic drugs in breast cancer cells. Thus, DDR1 signaling provides a novel target for therapeutic intervention with the prosurvival/antiapoptotic machinery of tumor cells.

HB-EGF is a potent inducer of tumor growth and angiogenesis.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors compared with normal tissue, its contribution to tumorigenicity is unknown. HB-EGF can be produced as a membrane-anchored form (pro-HB-EGF) and later processed to a soluble form (s-HB-EGF), although a significant amount of pro-HB-EGF remains uncleaved on the cell surface. To understand the roles of two forms of HB-EGF in promoting tumor growth, we have studied the effects of HB-EGF expression in the process of tumorigenesis using in vitro and in vivo systems. We demonstrate here that in EJ human bladder cancer cells containing a tetracycline-regulatable s-HB-EGF or pro-HB-EGF expression system, s-HB-EGF expression increased their transformed phenotypes, including growth rate, colony-forming ability, and activation of cyclin D1 promoter, as well as induction of vascular endothelial growth factor in vitro. Moreover, s-HB-EGF or wild-type HB-EGF induced the expression and activities of the metalloproteases, MMP-9 and MMP-3, leading to enhanced cell migration. In vivo studies also demonstrated that tumor cells expressing s-HB-EGF or wild-type HB-EGF significantly enhanced tumorigenic potential in athymic nude mice and exerted an angiogenic effect, increasing the density and size of tumor blood vessels. However, cells expressing solely pro-HB-EGF did not exhibit any significant tumorigenic potential. These findings establish s-HB-EGF as a potent inducer of tumor growth and angiogenesis and suggest that therapeutic intervention aimed at the inhibition of s-HB-EGF functions may be useful in cancer treatment.

Function of Drg1/Rit42 in p53-dependent mitotic spindle checkpoint.

Mutations in the Drg1/RTP/Rit42 gene are commonly identified in hereditary neuropathies of the motor and sensory systems. This gene was also identified as a p53 target gene and a differentiation-related, putative metastatic suppressor gene in human colon and prostate cancer. In this study, we show that the Rit42 protein is a microtubule-associated protein that localizes to the centrosomes and participates in the spindle checkpoint in a p53-dependent manner. When ectopically expressed and exposed to spindle inhibitors, Rit42 inhibited polyploidy in several p53-deficient tumor cell lines and increased the population of cells in mitotic arrest. Blocking endogenous Rit42 expression by small interfering RNA in normal human mammary epithelial cells resulted in the disappearance of astral microtubules, and dividing spindle fiber formation was rarely detected. Moreover, these cells underwent microtubule inhibitor-induced reduplication, leading to a polyploidy state. Our findings imply that Rit42 plays a role in the regulation of microtubule dynamics and the maintenance of euploidy.

BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition.

Aurora-A/BTAK/STK15 localizes to the centrosome in the G(2)-M phase, and its kinase activity regulates the G(2) to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G(2)-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314-1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser(308) of BRCA1 is phosphorylated by Aurora-A in vitro. Anti-phospho-specific antibodies against Ser(308) of BRCA1 demonstrated that Ser(308) is phosphorylated in vivo. Phosphorylation of Ser(308) increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser(308), and transient infection of adenovirus Aurora-A increased Ser(308) phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G(2) arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G(2) to M transition of cell cycle.

IFI16 as a negative regulator in the regulation of p53 and p21(Waf1).

IFI16 is a member of the HIN-200 family (hematopoietic interferon-inducible nuclear antigens with 200 amino acid repeats) that contains a DNA binding domain, a transcriptional regulatory domain, and DAPIN/PAAD, a protein domain associated with interferon response. It can function as a transcription repressor and directly binds p53. Although the structural and biochemical properties of IFI16 are known, the physiological relevance of these properties in the cellular context is still elusive. Here we report that the inhibition of endogenous IFI16 expression by small interfering RNA (siRNA) induces p21Waf1 mRNA and protein expression through p53 but does not induce pro-apoptotic p53 target genes. This rapid induction of p21 was wild-type p53-dependent and resulted in cell cycle arrest along with a marked reduction of phosphorylated Rb in normally growing cells. We also showed that the repression of IFI16 affects p53 transcriptional activity at the p21 promoter as well as the protein stability of p53 and p21. Our findings identified a new role for IFI16 in modulating p53 function and its target gene regulation in the control of cell cycle regulation.

BRCA1 shifts p53-mediated cellular outcomes towards irreversible growth arrest.

The tumor suppressor protein BRCA1 has been shown to enhance p53 transcription, whereas activated p53 represses BRCA1 transcription. To further understand the functional interaction of these proteins, we investigated the role of BRCA1 in p53-induced phenotypes. We found that BRCA1 when subjected to forced expression acts synergistically with wild-type p53, resulting in irreversible growth arrest, as shown by VhD mouse fibroblast cells expressing a temperature-sensitive mutant of p53. Furthermore, reintroduction of both BRCA1 and p53 into BRCA1(-/-)/p53(-/-) mouse embryonic fibroblasts markedly increased the senescence phenotype compared to that induced by p53 alone. In particular, we found that BRCA1 expression attenuated p53-mediated cell death in response to gamma-irradiation. Moreover, microarray screening of 11 000 murine genes demonstrated that a set of genes upregulated by p53 is enhanced by coexpression of BRCA1 and p53, suggesting that BRCA1 and p53 exert a promoter selectivity leading to a specific phenotype. Taken together, our results provide evidence that BRCA1 is involved in p53-mediated growth suppression rather than apoptosis.

p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop.

DDR1, discoidin domain receptor 1, belongs to a subfamily of tyrosine kinase receptors with an extracellular domain homologous to Dictyostellium discoideum protein discoidin 1. We showed that DDR1 is a direct p53 transcriptional target, and that DNA damage induced a p53-dependent DDR1 response associated with activation of its tyrosine kinase. We further demonstrated that DDR1 activated the MAPK cascade in a Ras-dependent manner. Whereas levels of p53, phosphoserine-15 p53, p21, ARF and Bcl-X(L) were increased in response to exogenous overexpression of activated DDR1, dominant-negative DDR1 inhibited irradiation-induced MAPK activation and p53, phosphoserine-15 p53, as well as induced p21 and DDR1 levels, suggesting that DDR1 functions in a feedforward loop to increase p53 levels and at least some of its effectors. Nonetheless, inhibition of DDR1 function resulted in strikingly increased apoptosis of wild-type p53-containing cells in response to genotoxic stress through a caspase-dependent pathway. These results strongly imply that this p53 response gene must predominately act to alleviate the adverse effects of stress induced by p53 on its target cell.