EMILIN1 role in tumor growth after enzymatic degradation

EMILIN1 is a ECM glycoprotein involved in several cellular processes. In particular, EMILIN1 is involved in elastogenesis processes and in the maintenance of lymphatic vessels structures. EMILIN1 presents several regulatory properties exercised through its EMI domain that is located at N-terminal region and is able to regulate homeostasis of blood pressure, and its gC1q domain located at C-terminal region. In particular, gC1q domain is involved in the regulation of both cell adhesion and cell proliferation through the binding with α4β1 integrin. EMILIN1-/- mice present an increase of tumoral cell proliferation; this is due to the loss of the interaction between gC1q domain-α4β1 integrin, that determines the activation of the MAPK pathway, resulting in upregulation of cell proliferation. Studies previously published in our laboratory demonstrated that neutrophil elastase, released by neutrophils present in tumor microenvironment, is able to degrade EMILIN1 resulting in the loss of its regulatory functions. Other authors have proposed EMILIN1 a possible substrate of several matrix metalloproteinases: matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase 14 (MT1-MMP). We demonstrated that among these MMPs only MT1-MMP shows a weak proteolytic activity on EMILIN1. Moreover, we observed that MT1-MMP was not able to impair EMILIN1 functions. On the contrary we observe that the digestion of EMILIN1 with neutrophil elastase was able to impair EMILIN1 tumor suppressor role. At this step, we wanted to analyze the capability of neutrophil elastase to degrade the gC1q domain. We digested the gC1q domain with several proteases and we observed that among the tested proteases only neutrophil elastase was able to degrade the gC1q domain and to impair its functionality. Thus, we wanted to pinpoint the neutrophil elastase cleavage site on gC1q domain, in order to generate a mutant of gC1q domain resistant to neutrophil elastase cleavage. We consulted several peptidase database that contained predicted neutrophile elastase cleavage sites, on these basis we generated several gC1q mutants. Among these mutants that generated, we found that the mutant R914W, in which aminoacid arginine was substituted with aminoacid tryptophan, was resistant to neutrophil elastase cleavage. Functional adhesion and proliferation assays confirmed the capability of R914 mutant to maintain its properties after neutrophil elastase treatment

EMILIN1 role in tumor growth after enzymatic degradation

EMILIN1 is a ECM glycoprotein involved in several cellular processes. In particular, EMILIN1 is involved in elastogenesis processes and in the maintenance of lymphatic vessels structures. EMILIN1 presents several regulatory properties exercised through its EMI domain that is located at N-terminal region and is able to regulate homeostasis of blood pressure, and its gC1q domain located at C-terminal region. In particular, gC1q domain is involved in the regulation of both cell adhesion and cell proliferation through the binding with α4β1 integrin. EMILIN1-/- mice present an increase of tumoral cell proliferation; this is due to the loss of the interaction between gC1q domain-α4β1 integrin, that determines the activation of the MAPK pathway, resulting in upregulation of cell proliferation. Studies previously published in our laboratory demonstrated that neutrophil elastase, released by neutrophils present in tumor microenvironment, is able to degrade EMILIN1 resulting in the loss of its regulatory functions. Other authors have proposed EMILIN1 a possible substrate of several matrix metalloproteinases: matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase 14 (MT1-MMP). We demonstrated that among these MMPs only MT1-MMP shows a weak proteolytic activity on EMILIN1. Moreover, we observed that MT1-MMP was not able to impair EMILIN1 functions. On the contrary we observe that the digestion of EMILIN1 with neutrophil elastase was able to impair EMILIN1 tumor suppressor role. At this step, we wanted to analyze the capability of neutrophil elastase to degrade the gC1q domain. We digested the gC1q domain with several proteases and we observed that among the tested proteases only neutrophil elastase was able to degrade the gC1q domain and to impair its functionality. Thus, we wanted to pinpoint the neutrophil elastase cleavage site on gC1q domain, in order to generate a mutant of gC1q domain resistant to neutrophil elastase cleavage. We consulted several peptidase database that contained predicted neutrophile elastase cleavage sites, on these basis we generated several gC1q mutants. Among these mutants that generated, we found that the mutant R914W, in which aminoacid arginine was substituted with aminoacid tryptophan, was resistant to neutrophil elastase cleavage. Functional adhesion and proliferation assays confirmed the capability of R914 mutant to maintain its properties after neutrophil elastase treatment.EMILIN1 è una glicoproteina della matrice extracellulare coinvolta in molti processi cellulari. Nella sua interezza è in grado di governare processi di elastogenesi dei tessuti e ha un ruolo importante nella regolazione della struttura dei vasi linfatici. Inoltre, è una proteina multi dominio, e, grazie ai suoi diversi domini funzionali, regola numerosi altri processi. Ad esempio, EMILIN1 regola l’omeostasi della pressione sanguigna tramite il suo dominio N-terminale, chiamato EMI domain; la regolazione dell’adesione e della proliferazione cellulare, invece, avviene tramite l’interazione tra il suo dominio C-terminale, chiamato gC1q, e l’integrina α4β1. L’effetto dell’interazione del dominio gC1q con l’integrina α4β1 è del tutto peculiare. Generalmente l’interazione delle molecole della matrice extracellulare con i recettori integrinici determina un aumento della proliferazione piuttosto una diminuizione come nel caso dell’interazione gC1q l’integrina α4β1. Studi condotti su topi EMILIN1-/- in cui non è possibile l’interazione gC1q-integrina α4β1, hanno messo in evidenza l’attivazione del pathway delle MAP chinasi, che induce un aumento della proliferazione cellulare. Dati precedentemente pubblicati nel nostro laboratorio hanno dimostrato che nel microambiente tumorale EMILIN1 viene degradata dalla elastasi rilasciata dai neutrofili, perdendo così le sue proprietà funzionali. Altri autori hanno ipotizzato, mediante l’uso di un approccio proteomico, che EMILIN1 può essere un substrato di alcune metalloproteasi, in particolare le metalloproteasi 3, 9 e 14. Nel presente lavoro si dimostra che queste tre metalloproteasi non sono in grado di svolgere un’ azione proteolitica rilevante. Tra queste tre metalloproteasi, infatti, soltanto la metalloproteasi 14 sembra esercitare un’azione proteolitica, anche se minima, su EMILIN1. Questa attività proteolitica comunque non è paragonabile a quella esercitata dall’elastasi neutrofila, ed inoltre, cosa ancora più importante, le proprietà funzionali di EMILIN1 non vengono compromesse dopo il trattamento della proteina con la metalloproteasi 14, come è stato dimostrato mediante saggi di adesione e proliferazione cellulare. Al contrario, il trattamento con l’elastasi neutrofila determina la perdita delle proprietà regolatorie di EMILIN1, causando una diminuzione dell’adesione ed un aumento della proliferazione cellulare e suggerendo che la degradazione avviene nel dominio gC1q. Tra gli enzimi testati, infatti, solo l’elastasi neutrofila è in grado di degradare e, quindi, compromettere le funzioni regolatorie del dominio funzionale gC1q. E’ nata, quindi, l’esigenza di identificare il sito/i di taglio dell’elastasi neutrofila sul dominio funzionale gC1, per poi, costruire un mutante resistente all’azione proteolitica dell’elastasi neutrofila. Consultando vari database riportanti i siti di taglio predetti sperimentalmente dell’elastasi neutrofila e mediante un approccio di mutagenesi sito-specifica, abbiamo creato vari mutanti. Tra questi un mutante che presentava la sostituzione dell’aminoacido arginina con l’aminoacido triptofano, il mutante R914W, si è dimostrato resistente all’azione proteolitica dell’elastasi neutrofila. Saggi funzionali di adesione e proliferazione hanno confermato la capacità, da parte di questo mutante, di preservare le sue proprietà regolatorie

Local inhibition of elastase reduces EMILIN1 cleavage reactivating lymphatic vessel function in a mouse lymphoedema model

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments

Somatostatin analogues increase AIP expression in somatotropinomas, irrespective of Gsp mutations

International audienceGermline aryl hydrocarbon receptor interacting protein (AIP) gene mutations confer a predisposition to pituitary adenoma (PA), predominantly GH-secreting (GH-PA). As recent data suggest a role for AIP in the pathogenesis of sporadic GH-PA and their response to somatostatin analogues (SSA), the expression of AIP and its partner, aryl hydrocarbon receptor (AHR), was determined by semiquantitative immunohistochemistry scoring in 62 sporadic GH-PA (37 treated with SSA preoperatively). The influence of Gsp status was studied in a subset of tumours (n=39, 14 Gsp(+)) and six GH-PA were available for primary cultures. AIP and AHR were detected in most cases, with a positive correlation between AIP and cytoplasmic AHR (P=0.012). Low AIP expression was significantly more frequent in untreated vs SSA-treated tumours (44.0 vs 20.5%, P=0.016). AHR expression or localisation did not differ between the two groups. Similarly, in vitro octreotide induced a median twofold increase in AIP expression (range 1.2-13.9, P=0.027) in GH-PA. In SSA-treated tumours, the AIP score was significantly higher in the presence of preoperative IGF1 decrease or tumour shrinkage (P=0.008 and P=0.014 respectively). In untreated tumours, low AIP expression was significantly associated with invasiveness (P=0.028) and suprasellar extension (P=0.019). The only effect of Gsp status was a significantly lower nuclear AHR score in Gsp(+) vs Gsp(-) tumours (P=0.025), irrespective of SSA. In conclusion, AIP is involved in the aggressiveness of sporadic GH-PA, regardless of Gsp status, and AIP up-regulation in SSA-treated tumours is associated with a better preoperative response, with no clear role for AHR