Mutant Ras and inflammation-driven skin tumorigenesis is suppressed via a JNK-iASPP-AP1 axis.

Concurrent mutation of a RAS oncogene and the tumor suppressor p53 is common in tumorigenesis, and inflammation can promote RAS-driven tumorigenesis without the need to mutate p53. Here, we show, using a well-established mutant RAS and an inflammation-driven mouse skin tumor model, that loss of the p53 inhibitor iASPP facilitates tumorigenesis. Specifically, iASPP regulates expression of a subset of p63 and AP1 targets, including genes involved in skin differentiation and inflammation, suggesting that loss of iASPP in keratinocytes supports a tumor-promoting inflammatory microenvironment. Mechanistically, JNK-mediated phosphorylation regulates iASPP function and inhibits iASPP binding with AP1 components, such as JUND, via PXXP/SH3 domain-mediated interaction. Our results uncover a JNK-iASPP-AP1 regulatory axis that is crucial for tissue homeostasis. We show that iASPP is a tumor suppressor and an AP1 coregulator.

Controversies around the function of LARP1.

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.

Cell autonomous role of iASPP deficiency in causing cardiocutaneous disorders.

Desmosome components are frequently mutated in cardiac and cutaneous disorders in animals and humans and enhanced inflammation is a common feature of these diseases. Previous studies showed that inhibitor of Apoptosis Stimulating p53 Protein (iASPP) regulates desmosome integrity at cell-cell junctions and transcription in the nucleus, and its deficiency causes cardiocutaneous disorder in mice, cattle, and humans. As iASPP is a ubiquitously expressed shuttling protein with multiple functions, a key question is whether the observed cardiocutaneous phenotypes are caused by loss of a cell autonomous role of iASPP in cardiomyocytes and keratinocytes specifically or by a loss of iASPP in other cell types such as immune cells. To address this, we developed cardiomyocyte-specific and keratinocyte-specific iASPP-deficient mouse models and show that the cell-type specific loss of iASPP in cardiomyocytes or keratinocytes is sufficient to induce cardiac or cutaneous disorders, respectively. Additionally, keratinocyte-specific iASPP-deficient mice have delayed eyelid development and wound healing. In keratinocytes, junctional iASPP is critical for stabilizing desmosomes and iASPP deficiency results in increased and disorganized cell migration, as well as impaired cell adhesion, consistent with delayed wound healing. The identification of a cell autonomous role of iASPP deficiency in causing cardiocutaneous syndrome, impaired eyelid development and wound healing suggests that variants in the iASPP gene also may contribute to polygenic heart and skin diseases.

Caspase cleavage of iASPP potentiates its ability to inhibit p53 and NF-κB.

An intriguing biological question relating to cell signaling is how the inflammatory mediator NF-kB and the tumour suppressor protein p53 can be induced by similar triggers, like DNA damage or infection, yet have seemingly opposing or sometimes cooperative biological functions. For example, the NF-κB subunit RelA/p65 has been shown to inhibit apoptosis, whereas p53 induces apoptosis. One potential explanation may be their co-regulation by common cellular factors: inhibitor of Apoptosis Stimulating p53 Protein (iASPP) is one such common regulator of both RelA/p65 and p53. Here we show that iASPP is a novel substrate of caspases in response to apoptotic stimuli. Caspase cleaves the N-terminal region of iASPP at SSLD294 resulting in a prominent 80kDa fragment of iASPP. This caspase cleavage site is conserved in various species from zebrafish to Homo sapiens. The 80kDa fragment of iASPP translocates from the cytoplasm to the nucleus via the RaDAR nuclear import pathway, independent of p53. The 80kDa iASPP fragment can bind and inhibit p53 or RelA/p65 more efficiently than full-length iASPP. Overall, these data reveal a potential novel regulation of p53 and RelA/p65 activities in response to apoptotic stimuli.

iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death.

Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

Emerin inhibits Lmo7 binding to the Pax3 and MyoD promoters and expression of myoblast proliferation genes.

X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) is caused by mutations in the inner nuclear membrane protein emerin. Previous studies have shown that emerin binds to and inhibits the activity of LIM domain only 7 (Lmo7), a transcription factor that regulates the expression of genes implicated in X-EDMD. Here, we analyzed Lmo7 function in C2C12 myoblast differentiation and its regulation by emerin. We found that Lmo7 was required for proper myoblast differentiation. Lmo7-downregulated myoblasts exhibited reduced expression of Pax3, Pax7, Myf5 and MyoD, whereas overexpression of GFP-Lmo7 increased the expression of MyoD and Myf5. Upon myotube formation, Lmo7 shuttled from the nucleus to the cytoplasm, concomitant with reduced expression of MyoD, Pax3 and Myf5. Importantly, we show that Lmo7 bound the Pax3, MyoD and Myf5 promoters both in C2C12 myoblasts and in vitro. Because emerin inhibited Lmo7 activity, we tested whether emerin competed with the MyoD promoter for binding to Lmo7 or whether emerin sequestered promoter-bound Lmo7 to the nuclear periphery. Supporting the competition model, emerin binding to Lmo7 inhibited Lmo7 binding to and activation of the MyoD and Pax3 promoters. These findings support the hypothesis that the functional interaction between emerin and Lmo7 is crucial for temporally regulating the expression of key myogenic differentiation genes.

Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA).

PURPOSE: Treatment of de-epithelialized human corneas with riboflavin (RF) + long-wavelength ultraviolet light (UVA; RFUVA) increases corneal stroma tensile strength significantly. RFUVA treatment retards the progression of keratoconus, perhaps by cross-linking of collagen molecules, but exact molecular mechanisms remain unknown. Research described here tested possible chemical mechanisms of cross-linking. METHODS: Corneas of rabbits and spiny dogfish sharks were de-epithelialized mechanically, subjected to various chemical pretreatments, exposed to RFUVA, and then subjected to destructive tensile stress measurements. Tensile strength was quantified with a digital force gauge to measure degree of tissue cross-linking. RESULTS: For both rabbit and shark corneas, RFUVA treatment causes significant cross-linking by mechanism(s) that can be blocked by the presence of sodium azide. Conversely, such cross-linking is greatly enhanced in the presence of deuterium oxide (D(2)O), even when RF is present at only one tenth the currently used clinical concentrations. Blocking carbonyl groups preexisting in the stroma with 2,4-dinitrophenylhydrazide or hydroxylamine blocks essentially all corneal cross-linking. In contrast, blocking free amine groups preexisting in the stroma with acetic anhydride or ethyl acetimidate does not affect RFUVA corneal cross-linking. When both carbonyl groups are blocked and singlet oxygen is quenched, no RFUVA cross-linking occurs, indicating the absence of other cross-linking mechanisms. CONCLUSIONS: RFUVA catalyzes cross-linking reactions that require production of singlet oxygen ((1)O(2)), whose half-life is extended by D(2)O. Carbonyl-based cross-linking reactions dominate in the corneal stroma, but other possible reaction schemes are proposed. The use of D(2)O as solution media for RF would enable concentration decreases or significant strength enhancement in treated corneas.