Aging enhances liver fibrotic response in mice through hampering extracellular matrix remodeling.

Clinical data identify age as a factor for severe liver fibrosis. We evaluate whether and how aging modulates the fibrotic response in a mouse model. Liver fibrosis was induced by CCl4 injections (thrice weekly for 2 weeks) in 7 weeks- and 15 months-old mice (young and old, respectively). Livers were analyzed for fibrosis, inflammation and remodeling 48 and 96 hours after the last injection. Old mice developed more severe fibrosis compared to young ones as evaluated by sirius red morphometry. Expression of pro-fibrogenic genes was equally induced in the two age-groups but enhanced fibrolysis in young mice was demonstrated by a significantly higher Mmp13 induction and collagenase activity. While fibrosis resolution occurred in young mice within 96 hours, no significant fibrosis attenuation was observed in old mice. Although recruitment of monocytes-derived macrophages was similar in young and old livers, young macrophages had globally a remodeling phenotype while old ones, a pro-fibrogenic phenotype. Moreover, we observed a higher proportion of thick fibers and enhanced expression of enzymes involved in collagen maturation in old mice. CONCLUSION: Impaired fibrolysis of a matrix less prone to remodeling associated with a pro-inflammatory phenotype of infiltrated macrophages contribute to a more severe fibrosis in old mice.

Regulation of matrix metalloproteinases activity studied in human endometrium as a paradigm of cyclic tissue breakdown and regeneration.

When abundant and activated, matrix metalloproteinases (MMPs, or matrixins) degrade most, if not all, constituents of the extracellular matrix (ECM). The resulting massive tissue breakdown is best exemplified in humans by the menstrual lysis and shedding of the endometrium, the mucosa lining the uterus. After menstruation, MMP activity needs to be tightly controlled as the endometrium regenerates and differentiates to avoid abnormal tissue breakdown while allowing tissue repair and fine remodelling to accommodate implantation of a blastocyst. This paper reviews how MMPs are massively present and activated in the endometrium at menstruation, and how their activity is tightly controlled at other phases of the cycle. Progesterone represses expression of many but not all MMPs. Its withdrawal triggers focal expression of MMPs specifically in the areas undergoing lysis, an effect mediated by local cytokines such as interleukin-1α, LEFTY-2, tumour necrosis factor-α and others. MMP-3 is selectively expressed at that time and activates proMMP-9, otherwise present in latent form throughout the cycle. In addition, a large number of neutrophils loaded with MMPs are recruited at menstruation through induction of chemokines, such as interleukin-8. At the secretory phase, progesterone repression of MMPs is mediated by transforming growth factor-ß. Tissue inhibitors of metalloproteinases (TIMPs) are abundant at all phases of the cycle to prevent any undue MMP activity, but are likely overwhelmed at menstruation. At other phases of the cycle, MMPs can elude TIMP inhibition as exemplified by recruitment of active MMP-7 to the plasma membrane of epithelial cells, allowing processing of membrane-associated growth factors needed for epithelial repair and proliferation. Finally, receptor-mediated endocytosis through low density lipoprotein receptor-related protein-1 (LRP-1) efficiently clears MMP-2 and -9 at the proliferative and secretory phases. This mechanism is probably essential to prevent any excessive ECM degradation by the active form of MMP-2 that is permanently present. However, shedding of the ectodomain of LRP-1 specifically at menstruation prevents endocytosis of MMPs allowing full degradation of the ECM. Thus endometrial MMPs are regulated at the levels of transcription, release from infiltrating neutrophils, activation, binding to the cell membrane, inhibition by TIMPs and endocytic clearance by LRP-1. This allows tight control during endometrial growth and differentiation but results in a burst of activity for menstrual tissue breakdown. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa α-chain) linked to a shorter transmembrane subunit (85-kDa ß-chain). LRP-1 cell-surface level and function are controlled by proteolytic shedding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ∼ 2-fold in cholesterol. Two membrane-associated metalloproteinases were involved in LRP-1 shedding: a disintegrin and metalloproteinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1-MMP, and specific tissue inhibitor of metalloproteinases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ∼4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by cholesterol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumulation of gelatinases (MMP-2 and MMP-9) in the medium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracellular proteolytic activities.

Post-translational proteolytic events influence LRP-1 functions.

The low-density lipoprotein receptor-related protein (LRP-1) is a membrane receptor displaying both endocytic scavenging and signaling functions. In this review, we briefly present post-translational proteolytic processes targeting this receptor and speculate on their possible influence on LRP-1 biological functions.

Metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 ectodomain decreases endocytic clearance of endometrial matrix metalloproteinase-2 and -9 at menstruation.

Cyclic elimination of the endometrium functional layer through menstrual bleeding results from intense tissue breakdown by proteolytic enzymes, mainly members of the matrix metalloproteinase (MMP) family. In contrast to menstrual-restricted MMPs, e.g. interstitial collagenase (MMP-1), gelatinases A (MMP-2) and B (MMP-9) mRNAs are abundant throughout the cycle without detectable tissue degradation at proliferative and secretory phases, implying a tight posttranslational control of both gelatinases. This paper addresses the role of low-density lipoprotein receptor-related protein (LRP)-1 in the endocytic clearance of endometrial gelatinases. LRP-1 mRNA and protein were studied using RT-PCR, Western blotting, and immunolabeling. Posttranslational control of LRP-1 was analyzed in explant culture. The receptor-associated protein (RAP), used as LRP antagonist, strongly increased (pro)gelatinase accumulation in medium conditioned by endometrial explants, suggesting a role for LRP-1 in their clearance. Although LRP-1 mRNA remained constant throughout the cycle, the protein ectodomain vanished at menses. LRP-1 immunolabeling selectively disappeared in areas of extracellular matrix breakdown in menstrual samples. It also disappeared from explants cultured without estrogen and progesterone (EP) due to ectodomain shedding in the medium. The shedding was inhibited by metalloproteinase inhibitors, including a disintegrin and metalloproteinase (ADAM) inhibitor, and by tissue inhibitors of MMPs (TIMP)-3 and -2, but barely by TIMP-1, pointing to ADAM-12 as the putative sheddase. In good agreement, ADAM-12 mRNA expression was repressed by EP. In conclusion, the efficient LRP-1-mediated clearance of gelatinase activity in nonbleeding endometrium is abrogated upon EP withdrawal, due to shedding of LRP-1 ectodomain by a metalloproteinase, presumably ADAM-12, itself regulated by EP.

Thyrotropin activates guanosine 5'-diphosphate/guanosine 5'-triphosphate exchange on the rate-limiting endocytic catalyst, Rab5a, in human thyrocytes in vivo and in vitro.

CONTEXT: We have previously reported that the TSH receptor/cAMP cascade enhances the coordinate expression of the rate-limiting endocytic catalysts, Rab5a and Rab7, which respectively promote thyroglobulin (Tg) internalization and transfer to lysosomes, thereby accelerating thyroid hormone secretion. OBJECTIVE: We address whether TSH further controls Rab5a activity by promoting its GTP-bound state. DESIGN: We compared Rab5a activation in seven pairs of hyperactive and corresponding quiescent thyroid tissues; TSH effect was reproduced on polarized cultures of normal human thyrocytes. PATIENTS: We studied seven euthyroid patients bearing hyperactive autonomous adenomas; normal thyroid tissue for culture. MAIN OUTCOME MEASUREMENTS: Rab5a GDP/GTP exchange factor activity [Rab5a-guanine nucleotide exchange factor (GEF)], expression of Rabex-5 (a Rab5a-GEF), and function of thyrocytes in vitro were the main outcome measures. RESULTS: In autonomous adenomas, constitutive activation increased both total activity and sedimentability (membrane recruitment) of Rab5a-GEF, compared with perinodular tissues. Increased Rab5a-GEF activity correlated with increased expression of Rabex-5 and Rab5a, as well as with Tg store depletion. In polarized human thyrocyte monolayers, TSH did not affect total Rab5a-GEF activity after 2 h but promoted its membrane recruitment; after 4 d, TSH increased both Rab5a-GEF activity and Rabex-5 expression and recruitment onto membranes where Rabex-5 coimmunoprecipitated with Rabaptin-5 and Rab5a. Sedimentable Rab5a-GEF perfectly correlated with apical endocytosis and lysosomal transfer of 125I-Tg, and with basolateral secretion of 125I-derived hormones. CONCLUSION: This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.