Differential Modulation of Matrix Metalloproteinases-2 and -7 in LAM/TSC Cells

Abstract

Matrix metalloproteinase (MMP) dysregulation is implicated in several diseases, given their involvement in extracellular matrix degradation and cell motility. In lymphangioleiomyomatosis (LAM), a pulmonary rare disease, MMP-2 and MMP-9 have been detected at high levels in serum and urine. LAM cells, characterized by a mutation in the tuberous sclerosis complex (TSC)1 or TSC2, promote cystic lung destruction. The role of MMPs in invasive and destructive LAM cell capability has not yet been fully understood. We evaluated MMP-2 and MMP-7 expression, secretion, and activity in primary LAM/TSC cells that bear a TSC2 germline mutation and an epigenetic modification and depend on epidermal growth factor (EGF) for survival. 5-azacytidine restored tuberin expression with a reduction of MMP-2 and MMP-7 levels and inhibits motility, similarly to rapamycin and anti-EGFR antibody. Both drugs reduced MMP-2 and MMP-7 secretion and activity during wound healing and decreased their expression in lung nodules of a LAM mouse model. In LAM/TSC cells, MMP-2 and MMP-7 are dependent on tuberin expression, cellular adhesion, and migration. MMPs appears sensitive to rapamycin and anti-EGFR antibody only during cellular migration. Our data indicate a complex and differential modulation of MMP-2 and MMP-7 in LAM/TSC cells, likely critical for lung parenchyma remodeling during LAM progression.

LAM Cells as Potential Drivers of Senescence in Lymphangioleiomyomatosis Microenvironment

“…In this case, the drug treatment caused cytotoxicity, which was not observed in our experimental conditions. We previously demonstrated that 5-azacytidine treatment also reduced migratory capability and MMP2 and MMP7 secretion of LAM/TSC cells, likely because of the induction of tuberin expression [11]. Given the broad inhibitory action of 5-azacytidine on hypermethylation that leads to chromatin remodelling, as often occurs in several types of cancers, we have to consider that this drug might cause other demethylating effects besides the induction of tuberin expression, making this point a limitation of our study that could be overcome by the retroviral transduction of TSC2.…”

“…At the state of the art, this hypothesis was only proposed but not demonstrated so far [27]. Indeed, it is already known that LAM cells exploit several mechanisms to induce the remodelling of the lung parenchyma, including the secretion of molecules such as matrix metalloproteinases [11], kathepsin K [10], VEGF-D [7], IL-6 [28], and IL-8 [16], which are also SASP components released by senescent cells to modulate the behaviour of neighbouring cells.…”

“…Western Blot (WB) analysis was performed as previously described [11]. Briefly, cells were lysed in lysis buffer (5 mM EDTA, 100 mM deoxycholic acid, 3% sodium dodecyl sulphate supplemented with protease inhibitors: Benzamidine 1 mM, PMSF 400 µM, Leupeptine 1 µg/mL, Aprotinin 10 µg/mL, Sigma-Aldrich), boiled for 5 min, and 30 µg of proteins for each sample were analysed by SDS-PAGE followed by the transfer to nitrocellulose membranes (Amersham, Arlington Height, IL, USA).…”

“…mTOR signalling is crucial for cell growth and proliferation since it induces cellular responses to stress, including the regulation of the metabolic activity and the contribution to the senescence establishment [9]. Recent studies demonstrated that LAM cells destroy the lung tissue by proteolytic activity through the secretion of cathepsin K [10] and metalloproteinases [11,12], whose secretion is also constitutively upregulated in SASP [3]. Similarly, the Vascular Endothelial Growth Factor-D (VEGF-D), a known biomarker for LAM [13], is a key component of SASP, being highly secreted from senescent fibroblasts to promote wound healing in human skin [14].…”

Primary TSC2-/meth Cells Induce Follicular Neogenesis in an Innovative TSC Mouse Model

“…Paraffin-embedded tissues were sectioned (thickness of 5 μm), deparaffinized, and rehydrated in xylene and decreasing concentrations of ethanol to distilled water (100%, 95%, 90%, 80%, and 70%). Immunohistochemistry analysis was performed as previously described [ 24 ]. Briefly, slides were treated under pressure at 95 °C in citrate buffer, pH 6.0 for 5 min for antigen retrieval, and incubated with 0.3% H 2 O 2 in methanol for 20 min to quench endogenous peroxidase activity.…”