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1.  Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1 
Biology of Reproduction  2011;86(3):78.
ABSTRACT
The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.
Expression of the metalloproteinase Mmp10 mRNA is stimulated by hCG prior to follicular rupture in both the human and the rat ovary, indicating involvement in ovulation and subsequent luteinization.
doi:10.1095/biolreprod.111.095588
PMCID: PMC3316269  PMID: 22116802
extracellular matrix; granulosa cells; matrix metalloproteinase; ovulation; ovulatory cycle; proteinases; theca cells
2.  Ovarian FAM110C (Family with Sequence Similarity 110C): Induction During the Periovulatory Period and Regulation of Granulosa Cell Cycle Kinetics in Rats1  
Biology of Reproduction  2012;86(6):185.
ABSTRACT
FAM110C belongs to a family of proteins that regulates cell proliferation. In the present study, the spatiotemporal expression pattern of FAM110C and its potential role were examined during the periovulatory period. Immature female rats were injected with equine chorionic gonadotropin (eCG) followed by human chorionic gonadotropin (hCG) and ovaries or granulosa cells were collected at various times after hCG administration (n = 3/time point). Expression levels of Fam110c mRNA and protein were highly induced both in intact ovaries and granulosa cells at 8 to 12 h after hCG treatment. In situ hybridization analysis demonstrated Fam110c mRNA expression was induced in theca and granulosa cells at 4 h after hCG, primarily localized to granulosa cells at 8 h and 12 h, and decreased at 24 h after hCG. There was negligible Fam110c mRNA detected in newly forming corpora lutea. In rat granulosa cell cultures, hCG induced expression of Fam110c mRNA was inhibited by RU486, whereas NS398 and AG1478 had no effect, suggesting that Fam110c expression is regulated in part by the progesterone receptor pathway. Promoter activity analysis revealed that an Sp1 site was important for the induction of Fam110c expression by hCG. Overexpression of FAM110C promoted granulosa cells to arrest at the G1 phase of the cell cycle but did not change progesterone levels. In summary, hCG induces Fam110c mRNA expression in granulosa cells by activation of an Sp1-binding site and the actions of progesterone. Our findings suggest that FAM110C may control granulosa cell differentiation into luteal cells by arresting cell cycle progression.
Human chorionic gonadotropin induces Fam110c mRNA expression in granulosa cells, which promotes their arrest at the G1 phase of the cell cycle; this suggests that FAM110C may control granulosa cell differentiation into luteal cells.
doi:10.1095/biolreprod.112.099259
PMCID: PMC3386148  PMID: 22460667
differentiation; granulosa cell; ovary; ovulation; progesterone; SP1
3.  Ovarian Furin (Proprotein Convertase Subtilisin/Kexin Type3): Expression, Localization, and Potential Role in Ovulation in the Rat1 
Biology of Reproduction  2010;83(1):147-154.
The process of ovulation involves weakening of the follicular wall by proteolytic enzymes. The function of FURIN (also known as PCSK3) is to activate various proteolytic enzymes. In the present study, the expression, localization, and function of FURIN were investigated in the periovulatory rat ovary. Immature female rats were injected with equine chorionic gonadotropin followed by human chorionic gonadotropin (hCG) 48 h later to stimulate ovulation. Ovaries were collected at 0, 4, 8, 12, and 24 h after hCG injection. Administration of hCG increased Furin mRNA expression in both intact ovaries and cultured ovarian follicles to maximal levels at 8 and 12 h before decreasing at 24 h. In cultured granulosa cells, Furin mRNA levels were significantly induced at 12 h after hCG. In situ hybridization of Furin mRNA demonstrated expression in the granulosa cells, with predominant expression in the theca layer. Regulation studies demonstrated that Furin mRNA was induced in residual tissue by forskolin or amphiregulin. To examine the role of FURIN in protease activation and ovulation, rats were treated with a FURIN inhibitor and oocyte release was determined. There was a 38% decrease in the number of oocytes released in ovaries treated with the FURIN inhibitor. Likewise, the FURIN inhibitor decreased the activation of MMP2. The induction of Furin mRNA after treatment with hCG, along with the decrease in MMP2 activation and oocyte release after FURIN inhibition, supports the hypothesis that FURIN is upregulated during the preovulatory period, which results in activation of proteinases associated with the breakdown of the follicular wall during ovulation.
Furin mRNA is upregulated by hCG prior to ovulation and FURIN inhibition blocks MMP2 activation and oocyte release.
doi:10.1095/biolreprod.109.079947
PMCID: PMC2888968  PMID: 20375258
follicle; ovary; ovulation; proteinase; theca cells
4.  Hormonal Induction of Polo-Like Kinases (Plks) and Impact of Plk2 on Cell Cycle Progression in the Rat Ovary 
PLoS ONE  2012;7(8):e41844.
The highly conserved polo-like kinases (Plks) are potent regulators of multiple functions in the cell cycle before and during mitotic cell division. We investigated the expression pattern of Plk genes and their potential role(s) in the rat ovary during the periovulatory period. Plk2 and Plk3 were highly induced both in intact ovaries and granulosa cells in vivo after treatment with the luteinizing hormone (LH) agonist, human chorionic gonadotropin (hCG). In vitro, hCG stimulated the expression of Plk2 in granulosa cells, but not Plk3. This induction of Plk2 expression was mimicked by both forskolin and phorbol 12 myristate 13-acetate (PMA). Moreover, Plk2 expression was reduced by inhibitors of prostaglandin synthesis or the EGF pathway, but not by progesterone receptor antagonist (RU486) treatment. At the promoter level, mutation of the Sp1 binding sequence abolished the transcriptional activity of the Plk2 gene. ChIP assays also revealed the interaction of endogenous Sp1 protein in the Plk2 promoter region. Functionally, the over-expression of Plk2 and Plk3 arrested granulosa cells at the G0/G1 phase of the cell cycle. In contrast, the knockdown of Plk2 expression in granulosa cells decreased the number of cells in the G0/G1 stage of the cell cycle, but increased granulosa cell viability. In summary, hCG induced Plk2 and Plk3 expression in the rat ovary. Prostaglandins and the EGF signaling pathway are involved in regulating Plk2 expression. The transcription factor Sp1 is important for Plk2 transcriptional up-regulation. Our findings suggest that the increase in Plk2 and Plk3 expression contributes to the cell cycle arrest of granulosa cells which is important for the luteinization of granulosa cells during the periovulatory period.
doi:10.1371/journal.pone.0041844
PMCID: PMC3411565  PMID: 22870256
5.  Inhibition of Gelatinase Activity Reduces Neural Injury in an Ex-Vivo Model of Hypoxia-Ischemia 
Neuroscience  2009;160(4):755-766.
Perinatal hypoxia-ischemia (H-I) often manifests as cognitive and/or motor disturbances that appear early in development. Growing evidence indicates that neuroinflammation may exacerbate H-I injury. Resident microglia release proinflammatory cytokines and proteases in response to ischemia. Matrix metalloproteinases (MMPs), in particular, activate cytokines and degrade basement membrane proteins. These actions ultimately permit entry of peripheral leukocytes into the central nervous system neuropil, enhancing neuroinflammation and cell death. Currently, the relative contributions of resident and peripheral immune cells to ischemic brain injury are unclear. The present study employed an ex-vivo model of H-I through oxygen glucose deprivation (OGD) to identify the cellular localization of MMP-9 in organotypic hippocampal slices, and to determine whether inhibiting gelatin-degrading MMPs affords neuroprotection in the absence of peripheral immune cells. Immunohistochemistry revealed ubiquitous neuronal MMP-9 expression in both normoxic and hypoxic slices. Increased MMP-9 expression was detected in CD11b – positive microglia after 48 hrs exposure to OGD relative to normoxic controls. Consistent with these data, in situ zymography showed increased gelatinolytic activity after OGD. Gelatin-cleaved fluorescence localized to astrocytic processes and somata of various cellular morphologies. Treatment with either the MMP inhibitor AG3340 (prinomastat) or minocycline dampened OGD – induced gelatinolytic activity and neural injury, as measured by Fluoro-Jade staining, relative to vehicle controls. These results show that resident microglia, in the absence of peripheral immune cells, were sufficient to enhance neural injury after OGD in the organotypic hippocampal slice. Additionally, these effects were associated with upregulation or secretion of MMP-9, and were blocked after treatment with either the gelatinase-selective compound AG3340 or the anti-inflammatory compound minocycline. These data, coupled with the effectiveness of these compounds previously shown in vivo, support the selective targeting of gelatin-degrading MMPs and activated microglia as potential therapeutic approaches to combat neonatal H-I injury.
doi:10.1016/j.neuroscience.2009.02.080
PMCID: PMC2746919  PMID: 19272421
inflammation; neuroprotection; central nervous system; matrix metalloproteinase; microglia; oxygen glucose deprivation
6.  Luteinizing Hormone-Induced RUNX1 Regulates the Expression of Genes in Granulosa Cells of Rat Periovulatory Follicles 
The LH surge induces specific transcription factors that regulate the expression of a myriad of genes in periovulatory follicles to bring about ovulation and luteinization. The present study determined 1) the localization of RUNX1, a nuclear transcription factor, 2) regulation of Runx1 mRNA expression, and 3) its potential function in rat ovaries. Up-regulation of mRNA and protein for RUNX1 is detected in preovulatory follicles after human chorionic gonadotropin (hCG) injection in gonadotropin-treated immature rats as well as after the LH surge in cycling animals by in situ hybridization and immunohistochemical and Western blot analyses. The regulation of Runx1 mRNA expression was investigated in vitro using granulosa cells from rat pre-ovulatory ovaries. Treatments with hCG, forskolin, or phorbol 12 myristate 13-acetate stimulated Runx1 mRNA expression. The effects of hCG were reduced by inhibitors of protein kinase A, MAPK kinase, or p38 kinase, indicating that Runx1 expression is regulated by the LH-initiated activation of these signaling mediators. In addition, hCG-induced Runx1 mRNA expression was inhibited by a progesterone receptor antagonist and an epidermal growth factor receptor tyrosine kinase inhibitor, whereas amphiregulin stimulated Runx1 mRNA expression, demonstrating that the expression is mediated by the activation of the progesterone receptor and epidermal growth factor receptor. Finally, knockdown of Runx1 mRNA by small interfering RNA decreased progesterone secretion and reduced levels of mRNA for Cyp11a1, Hapln1, Mt1a, and Rgc32. The hormonally regulated expression of Runx1 in periovulatory follicles, its involvement in progesterone production, and regulation of preovulatory gene expression suggest important roles of RUNX1 in the periovulatory process.
doi:10.1210/me.2005-0512
PMCID: PMC1783681  PMID: 16675540
AML1, Acute myeloid leukemia 1; AREG, amphiregulin; cdkn, cyclin-dependent kinase inhibitor; C/EBPβ, CCAAT-enhancer binding protein β; CG, chorionic gonadotropin; DMSO, dimethylsulfoxide; EGF, epidermal growth factor; Hapln1, hyaluronan and proteoglycan link protein 1; MEK, MAPK kinase; Mt1a, metallothionein 1a; PGR, progesterone receptor; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12 myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; Rgc32, response gene to complement 32; siRNA, small interfering RNA; Timp1, tissue inhibitor of metalloproteinase-1
7.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
8.  Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin 
British Journal of Cancer  2000;82(3):657-665.
Tissue from 54 histologically-identified basal cell carcinomas of the skin was obtained at surgery and assayed using a combination of functional and immunochemical procedures for matrix metalloproteinases (MMPs) with collagenolytic activity and for MMPs with gelatinolytic activity. Collagenolytic enzymes included MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3). Gelatinolytic enzymes included MMP-2 (72-kDa gelatinase A/type IV collagenase) and MMP-9 (92-kDa gelatinase B/type IV collagenase). Inhibitors of MMP activity including tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) were also assessed. All three collagenases and both gelatinases were detected immunochemically. MMP-1 appeared to be responsible for most of the functional collagenolytic activity while gelatinolytic activity reflected both MMP-2 and MMP-9. MMP inhibitor activity was also present, and appeared, based on immunochemical procedures, to reflect the presence of TIMP-1 but not TIMP-2. As a group, tumours identified as having aggressive-growth histologic patterns were not distinguishable from basal cell carcinomas with less aggressive-growth histologic patterns. In normal skin, the same MMPs were detected by immunochemical means. However, only low to undetectable levels of collagenolytic and gelatinolytic activities were present. In contrast, MMP inhibitor activity was comparable to that seen in tumour tissue. In previous studies we have shown that exposure of normal skin to epidermal growth factor in organ culture induces MMP up-regulation and activation. This treatment concomitantly induces stromal invasion by the epithelium (Varani et al (1995) Am J Pathol146: 210–217; Zeigler et al (1996 b) Invasion Metastasis16: 11–18). Taken together with these previous data, the present findings allow us to conclude that the same profile of MMP/MMP inhibitors that is associated with stromal invasion in the organ culture model is expressed endogenously in basal cell carcinomas of skin. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.1999.0978
PMCID: PMC2363319  PMID: 10682680
interstitial collagenase; collagenase-3; tissue inhibitoral metalloproteinase invasion; fibroblast; epithelial cells; endothelial cells
9.  Prostaglandin E2 Receptors Are Differentially Expressed in Subpopulations of Granulosa Cells from Primate Periovulatory Follicles1 
Biology of Reproduction  2011;85(5):916-923.
Prostaglandin E2 (PGE2) mediates many effects of the midcycle luteinizing hormone (LH) surge within the periovulatory follicle. Differential expression of the four PGE2 (EP) receptors may contribute to the specialized functions of each granulosa cell subpopulation. To determine if EP receptors are differentially expressed in granulosa cells, monkeys received gonadotropins to stimulate ovarian follicular development. Periovulatory events were initiated with human chorionic gonadotropin (hCG); granulosa cells and whole ovaries were collected before (0 h) and after (24–36 h) hCG to span the 40-h primate periovulatory interval. EP receptor mRNA and protein levels were quantified in granulosa cell subpopulations. Cumulus cells expressed higher levels of EP2 and EP3 mRNA compared with mural cells 36 h after hCG. Cumulus cell EP2 and EP3 protein levels also increased between 0 and 36 h after hCG. Overall, mural granulosa cells expressed low levels of EP1 protein at 0 h and higher levels 24–36 h after hCG. However, EP1 protein levels were higher in granulosa cells away from the follicle apex compared with apex cells 36 h after hCG. Higher levels of PAI-1 protein were measured in nonapex cells, consistent with a previous study showing EP1-stimulated PAI-1 protein expression in monkey granulosa cells. EP4 protein levels were low in all subpopulations. In summary, cumulus cells likely respond to PGE2 via EP2 and EP3, whereas PGE2 controls rupture of a specific region of the follicle via EP1. Therefore, differential expression of EP receptors may permit each granulosa cell subpopulation to generate a unique response to PGE2 during the process of ovulation.
Each of four PGE2 receptors is differentially expressed in subpopulations of granulosa cells, suggesting each subpopulation may generate a unique response to PGE2 during ovulation.
doi:10.1095/biolreprod.111.091306
PMCID: PMC3197913  PMID: 21753194
cumulus cells; granulosa cells; hormone receptors; ovulation; prostaglandins
10.  Regulated Expression of Rhox8 in the Mouse Ovary: Evidence for the Role of Progesterone and RHOX5 in Granulosa Cells1 
Biology of Reproduction  2013;88(5):126.
ABSTRACT
The gonadotropin surge is the essential trigger to stimulate ovulation and luteinization of ovarian follicles. While the hormone signals from the brain that initiate ovulation are known, the specific targets which regulate this process are not well known. In this study, we assessed the suitability of the Rhox homeobox gene cluster to serve as the master regulators of folliculogenesis. In superovulated (equine chorionic gonadotropin [eCG]/human chorionic gonadotropin [hCG]) mice, the Rhox genes exhibited four distinct windows of peak expression, suggesting that these genes may regulate specific events during the ovulatory cycle. Like many members of the cluster, Rhox8 mRNA and protein were induced by follicle stimulating hormone [FSH]/eCG in granulosa cells. However, Rhox8 displayed unique peak expression at 8 h post-hCG administration, implying it might be the lone member of the cluster regulated by progesterone. Subsequent promoter analysis in granulosa cells revealed relevant homeobox binding and progesterone response elements within Rhox8's 5′-flanking region. In superovulated mice, progesterone receptor (PGR) is recruited to the Rhox8 promoter, as assessed by chromatin immunoprecipitation. In Rhox5-null mice, Rhox8 mRNA was reduced at 2 h and 4 h post-hCG administration but recovered once the follicles passed the antral stage of development. Conversely, in progesterone receptor knockout mice, Rhox8 exhibited normal stimulation by eCG but failed to reach its peak mRNA level at 8 h post-hCG found in wild-type mice. This suggests a model in which Rhox8 transcription is dependent upon RHOX5 during early folliculogenesis and upon progesterone during the periovulatory window when RHOX5 normally wanes. In support of this model, transfection of RHOX5 and PGR expression plasmids stimulated, whereas dominant negative and mutant constructs inhibited, Rhox8 promoter activity.
The Rhox homeobox gene cluster is differentially expressed during folliculogenesis in mice; RHOX8 is a potential candidate for translating progesterone signaling into successful ovulation.
doi:10.1095/biolreprod.112.103267
PMCID: PMC4013910  PMID: 23536368
gene regulation; granulosa cells; progesterone; progesterone receptor; transcriptional regulation
11.  Two Pathways for Prostaglandin F2α (PGF2α) Synthesis by the Primate Periovulatory Follicle 
Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2α levels in primate periovulatory follicles. To better understand the role of PGF2α in ovulation, pathways utilized for PGF2α synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40-hour periovulatory interval. Human granulosa cells were also obtained (typically 34-36 hours after hCG) from in vitro fertilization patients. PGF2α can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24-36 hours after hCG administration. Human granulosa cells converted PGD2 into 11β-PGF2α, confirming that these cells possess AKR1C3 activity. PGF2α can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0-12 hours, peaked at 24 hours, and returned to low levels by 36 hours after hCG administration. Human granulosa cell conversion of 3H-PGE2 into 3H-PGF2α was reduced by a AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2α can be synthesized via two pathways during the periovulatory interval.
doi:10.1530/REP-07-0514
PMCID: PMC2656351  PMID: 18390687
ovulation; ovary; prostaglandin; granulosa cell; monkey; primate
12.  Fine-structural distribution of MMP-2 and MMP-9 activities in the rat skeletal muscle upon training: a study by high-resolution in situ zymography 
Histochemistry and Cell Biology  2012;138(1):75-87.
Matrix metalloproteinases (MMPs) are key regulators of extracellular matrix remodeling, but have also important intracellular targets. The purpose of this study was to examine the activity and subcellular localization of the gelatinases MMP-2 and MMP-9 in skeletal muscle of control and physically trained rats. In control hind limb muscle, the activity of the gelatinases was barely detectable. In contrast, after 5 days of intense exercise, in Soleus (Sol), but not Extensor digitorum longus (EDL) muscle, significant upregulation of gelatinolytic activity in myofibers was observed mainly in the nuclei, as assessed by high resolution in situ zymography. The nuclei of quiescent satellite cells did not contain the activity. Within the myonuclei, the gelatinolytic activity colocalized with an activated RNA Polymerase II. Also in Sol, but not in EDL, there were few foci of mononuclear cells with strongly positive cytoplasm, associated with apparent necrotic myofibers. These cells were identified as activated satellite cells/myoblasts. No extracellular gelatinase activity was observed. Gel zymography combined with subcellular fractionation revealed training-related upregulation of active MMP-2 in the nuclear fraction, and increase of active MMP-9 in the cytoplasmic fraction of Sol. Using RT-PCR, selective increase in MMP-9 mRNA was observed. We conclude that training activates nuclear MMP-2, and increases expression and activity of cytoplasmic MMP-9 in Sol, but not in EDL. Our results suggest that the gelatinases are involved in muscle adaptation to training, and that MMP-2 may play a novel role in myonuclear functions.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-012-0940-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-012-0940-5
PMCID: PMC3374103  PMID: 22419075
Matrix metalloproteinase; Exercise; Skeletal muscle; Rat; Cell nucleus; Satellite cells
13.  Identification and characterization of small-molecule inhibitors of hepsin 
Molecular cancer therapeutics  2008;7(10):3343-3351.
Hepsin is a type-II transmembrane serine protease overexpressed in the majority of human prostate cancers. We recently demonstrated that hepsin promotes prostate cancer progression and metastasis and thus represents a potential therapeutic target. Here we report the identification of novel small-molecule inhibitors of hepsin catalytic activity. We utilized purified human hepsin for high-throughput screening of established drug and chemical diversity libraries and identified sixteen inhibitory compounds with IC50 values against hepsin ranging from 0.23–2.31μM and relative selectivity of up to 86-fold or greater. Two compounds are orally administered drugs established for human use. Four compounds attenuated hepsin-dependent pericellular serine protease activity in a dose dependent manner with limited or no cytotoxicity to a range of cell types. These compounds may be used as leads to develop even more potent and specific inhibitors of hepsin to prevent prostate cancer progression and metastasis.
doi:10.1158/1535-7163.MCT-08-0446
PMCID: PMC2659609  PMID: 18852137
hepsin; prostate; metastasis; inhibitor; cancer
14.  Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues 
British Journal of Cancer  2005;92(12):2171-2180.
Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT–PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.
doi:10.1038/sj.bjc.6602630
PMCID: PMC2361819  PMID: 15928670
matrix metalloproteinases; serine proteases; prostate cancer
15.  Effect of Antiprogesterone RU486 on VEGF Expression and Blood Vessel Remodeling on Ovarian Follicles before Ovulation 
PLoS ONE  2014;9(4):e95910.
Background
The success of ovarian follicle growth and ovulation is strictly related to the development of an adequate blood vessel network required to sustain the proliferative and endocrine functions of the follicular cells. Even if the Vascular Endothelial Growth Factor (VEGF) drives angiogenesis before ovulation, the local role exerted by Progesterone (P4) remains to be clarified, in particular when its concentration rapidly increases before ovulation.
Aim
This in vivo study was designed to clarify the effect promoted by a P4 receptor antagonist, RU486, on VEGF expression and follicular angiogenesis before ovulation, in particular, during the transition from pre to periovulatory follicles induced by human Chorionic Gonadotropins (hCG) administration.
Material and Methods
Preovulatory follicle growth and ovulation were pharmacologically induced in prepubertal gilts by combining equine Chorionic Gonadotropins (eCG) and hCG used in the presence or absence of RU486. The effects on VEGF expression were analyzed using biochemical and immunohistochemical studies, either on granulosa or on theca layers of follicles isolated few hours before ovulation. This angiogenic factor was also correlated to follicular morphology and to blood vessels architecture.
Results and Conclusions
VEGF production, blood vessel network and follicle remodeling were impaired by RU486 treatment, even if the cause-effect correlation remains to be clarified. The P4 antagonist strongly down-regulated theca VEGF expression, thus, preventing most of the angiogenic follicle response induced by hCG. RU486-treated follicles displayed a reduced vascular area, a lower rate of endothelial cell proliferation and a reduced recruitment of perivascular mural cells. These data provide important insights on the biological role of RU486 and, indirectly, on steroid hormones during periovulatory follicular phase. In addition, an in vivo model is proposed to evaluate how periovulatory follicular angiogenesis may affect the functionality of the corpus luteum (CL) and the success of pregnancy.
doi:10.1371/journal.pone.0095910
PMCID: PMC3995877  PMID: 24756033
16.  Activation and localization of matrix metalloproteinase-2 and -9 in the skeletal muscle of the muscular dystrophy dog (CXMDJ) 
Background
Matrix metalloproteinases (MMPs) are key regulatory molecules in the formation, remodeling and degradation of all extracellular matrix (ECM) components in both physiological and pathological processes in various tissues. The aim of this study was to examine the involvement of gelatinase MMP family members, MMP-2 and MMP-9, in dystrophin-deficient skeletal muscle. Towards this aim, we made use of the canine X-linked muscular dystrophy in Japan (CXMDJ) model, a suitable animal model for Duchenne muscular dystrophy.
Methods
We used surgically biopsied tibialis cranialis muscles of normal male dogs (n = 3) and CXMDJ dogs (n = 3) at 4, 5 and 6 months of age. Muscle sections were analyzed by conventional morphological methods and in situ zymography to identify the localization of MMP-2 and MMP-9. MMP-2 and MMP-9 activity was examined by gelatin zymography and the levels of the respective mRNAs in addition to those of regulatory molecules, including MT1-MMP, TIMP-1, TIMP-2, and RECK, were analyzed by semi-quantitative RT-PCR.
Results
In CXMDJ skeletal muscle, multiple foci of both degenerating and regenerating muscle fibers were associated with gelatinolytic MMP activity derived from MMP-2 and/or MMP-9. In CXMDJ muscle, MMP-9 immunoreactivity localized to degenerated fibers with inflammatory cells. Weak and disconnected immunoreactivity of basal lamina components was seen in MMP-9-immunoreactive necrotic fibers of CXMDJ muscle. Gelatinolytic MMP activity observed in the endomysium of groups of regenerating fibers in CXMDJ did not co-localize with MMP-9 immunoreactivity, suggesting that it was due to the presence of MMP-2. We observed increased activities of pro MMP-2, MMP-2 and pro MMP-9, and levels of the mRNAs encoding MMP-2, MMP-9 and the regulatory molecules, MT1-MMP, TIMP-1, TIMP-2, and RECK in the skeletal muscle of CXMDJ dogs compared to the levels observed in normal controls.
Conclusion
MMP-2 and MMP-9 are likely involved in the pathology of dystrophin-deficient skeletal muscle. MMP-9 may be involved predominantly in the inflammatory process during muscle degeneration. In contrast, MMP-2, which was activated in the endomysium of groups of regenerating fibers, may be associated with ECM remodeling during muscle regeneration and fiber growth.
doi:10.1186/1471-2474-8-54
PMCID: PMC1929071  PMID: 17598883
17.  Role of hypoxia in the regulation of periovulatory endothelin-2 (EDN2) expression in the mouse 
Endothelin-2 (EDN2) was recently proposed as a granulosa cell-derived contractile signal that facilitates ovulation (Ko et al., Endocrinology 147(4):1770-1779, 2006). Spatially, Edn2 mRNA expression is restricted to granulosa cells of periovulatory follicles. Temporally, mRNA for this contractile peptide is expressed immediately prior to follicle rupture. The primary objective of this study was to test the hypothesis that hypoxia mediates END2 expression in granulosa cells at ovulation and if so, to determine the region within the EDN2 promoter responsible for this effect. To determine the effect of hypoxia on Edn2 mRNA expression, immature mice were treated with 5 IU PMSG followed 48 h later by 5 IU hCG. Granulosa cells were isolated at 9 h after hCG, cultured under normal or hypoxic conditions and the expression level of mRNA for Edn2 was compared. Edn2 mRNA expression was increased when granulosa cells were cultured in a hypoxic environment (p < 0.05). Subsequent promoter analysis found that the 5′upstream region of the EDN2 promoter (between -1894 and -1407 bp) was responsible for hypoxia-mediated changes in EDN2 expression. This promoter region contains multiple sites for potential transcriptional regulation including that by hypoxia-inducible factor 1 (HIF-1; ACGTG) at -1297 bp. The second objective of this study was to determine whether the progesterone receptor (PR) or cyclooxygenase-2 (COX-2), two key regulators of periovulatory events, controlled EDN2 expression. To accomplish this, gonadotropin-primed mice were treated with RU-486 or indomethacin and expression of mRNA for Edn2 was determined in ovaries collected at 12 h after hCG. Treatment with RU-486 or indomethacin did not affect expression of mRNA for Edn2 (p > 0.05). Taken together, it is believed that hypoxia, but not the PR or COX-2, regulate gonadotropin-induced EDN2 expression in the periovulatory follicle.
doi:10.1139/y08-025
PMCID: PMC3027409  PMID: 18516093
endothelin-2; ovulation; ovary; hypoxia
18.  Hormonal Regulation of MicroRNA Expression in Periovulatory Mouse Mural Granulosa Cells1 
Biology of Reproduction  2008;79(6):1030-1037.
MicroRNAs (miRNAs) mediate posttranscriptional gene regulation by binding to the 3′ untranslated region of messenger RNAs to either inhibit or enhance translation. The extent and hormonal regulation of miRNA expression by ovarian granulosa cells and their role in ovulation and luteinization is unknown. In the present study, miRNA array analysis was used to identify 212 mature miRNAs as expressed and 13 as differentially expressed in periovulatory granulosa cells collected before and after an ovulatory dose of hCG. Two miRNAs, Mirn132 and Mirn212 (also known as miR-132 and miR-212), were found to be highly upregulated following LH/hCG induction and were further analyzed. In vivo and in vitro temporal expression analysis by quantitative RT-PCR confirmed that LH/hCG and cAMP, respectively, increased transcription of the precursor transcript as well as the mature miRNAs. Locked nucleic acid oligonucleotides complementary to Mirn132 and Mirn212 were shown to block cAMP-mediated mature miRNA expression and function. Computational analyses indicated that 77 putative mRNA targets of Mirn132 and Mirn212 were expressed in ovarian granulosa cells. Furthermore, upon knockdown of Mirn132 and Mirn212, a known target of Mirn132, C-terminal binding protein 1, showed decreased protein levels but no change in mRNA levels. The following studies are the first to describe the extent of miRNA expression within ovarian granulosa cells and the first to demonstrate that LH/hCG regulates the expression of select miRNAs, which affect posttranscriptional gene regulation within these cells.
The ovulatory surge of luteinizing hormone induces the expression of microRNAs, which posttranscriptionally regulate the expression of a regulatory protein within mural granulosa cells of the periovulatory follicle.
doi:10.1095/biolreprod.108.069690
PMCID: PMC2780477  PMID: 18716288
corpus luteum; granulosa cells; luteinizing hormone; microRNA; ovulation
19.  Doxycycline-mediated Inhibition of Choroidal Neovascularization 
Purpose
Doxycycline, a broad spectrum antibiotic, has certain anti-angiogenic properties and can inhibit matrix metalloproteinases (MMPs/gelatinases). We investigated the effects of doxycycline on choroidal neovascularization (CNV), and regulation of MMP-2/-9 and pigment epithelium-derived factor (PEDF).
Methods
Doxycycline was orally administered to rats at 500, 50, 5, and 0.5 mg/kg/day, using non-treated animals as controls. Experimental CNV was induced with laser 7 days after doxycycline treatment started. At seven days post-induction, animals were euthanized, and eyes collected. RPE/choroid flat-mounts were labeled with isolectin IB4 to determine CNV lesion volumes using confocal microscopy and Volocity® software. MMP-2, MMP-9 and PEDF protein levels were determined by ELISA. MMP catalytic activity was determined in solution using fluorogenic gelatin and peptide substrates, by gelatin zymography in SDS-PAGE and by in situ DQ-gelatin zymography in RPE/choroid sections.
Results
CNV complex lesion volumes decreased with doxycycline in a dose-response relationship. A dosage of 500 mg/kg/day caused a 70% inhibition of CNV complex volume compared to control animals. Doxycycline elevated PEDF levels in plasma, and did not affect the plasma pro- and active MMP-2 and MMP-9 levels. However, the in vitro enzymatic activities of purified MMP-2 and MMP-9 declined significantly with doxycycline. MMP-2, MMP-9 and gelatinolytic activities in situ increased early in CNV lesion development. Doxycycline treatments and exogenous additions inhibited gelatinolytic activities in CNV lesions.
Conclusions
Doxycycline effectively hampered the progression of experimental CNV. The results suggest that orally administrated doxycycline can reach the choroid to attenuate proteolytic enzymes that remodel Bruch's membrane and promote the anti-angiogenic PEDF to inhibit neovascularization.
doi:10.1167/iovs.08-3174
PMCID: PMC2836119  PMID: 19516001
20.  Pharmacoproteomics of a Metalloproteinase Hydroxamate Inhibitor in Breast Cancer Cells: Dynamics of Membrane Type 1 Matrix Metalloproteinase-Mediated Membrane Protein Shedding ▿ ‡  
Molecular and Cellular Biology  2008;28(15):4896-4914.
Broad-spectrum matrix metalloproteinase (MMP) inhibitors (MMPI) were unsuccessful in cancer clinical trials, partly due to side effects resulting from limited knowledge of the full repertoire of MMP substrates, termed the substrate degradome, and hence the in vivo functions of MMPs. To gain further insight into the degradome of MMP-14 (membrane type 1 MMP) an MMPI, prinomastat (drug code AG3340), was used to reduce proteolytic processing and ectodomain shedding in human MDA-MB-231 breast cancer cells transfected with MMP-14. We report a quantitative proteomic evaluation of the targets and effects of the inhibitor in this cell-based system. Proteins in cell-conditioned medium (the secretome) and membrane fractions with levels that were modulated by the MMPI were identified by isotope-coded affinity tag (ICAT) labeling and tandem mass spectrometry. Comparisons of the expression of MMP-14 with that of a vector control resulted in increased MMP-14/vector ICAT ratios for many proteins in conditioned medium, indicating MMP-14-mediated ectodomain shedding. Following MMPI treatment, the MMPI/vehicle ICAT ratio was reversed, suggesting that MMP-14-mediated shedding of these proteins was blocked by the inhibitor. The reduction in shedding or the release of substrates from pericellular sites in the presence of the MMPI was frequently accompanied by the accumulation of the protein in the plasma membrane, as indicated by high MMPI/vehicle ICAT ratios. Considered together, this is a strong predictor of biologically relevant substrates cleaved in the cellular context that led to the identification of many undescribed MMP-14 substrates, 20 of which we validated biochemically, including DJ-1, galectin-1, Hsp90α, pentraxin 3, progranulin, Cyr61, peptidyl-prolyl cis-trans isomerase A, and dickkopf-1. Other proteins with altered levels, such as Kunitz-type protease inhibitor 1 and beta-2-microglobulin, were not substrates in biochemical assays, suggesting an indirect affect of the MMPI, which might be important in drug development as biomarkers or, in preclinical phases, to predict systemic drug actions and adverse side effects. Hence, this approach describes the dynamic pattern of cell membrane ectodomain shedding and its perturbation upon metalloproteinase drug treatment.
doi:10.1128/MCB.01775-07
PMCID: PMC2493375  PMID: 18505826
21.  Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. 
Journal of Clinical Investigation  1994;94(6):2493-2503.
Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.
Images
PMCID: PMC330083  PMID: 7989608
22.  Methods for Analysis of Matrix Metalloproteinase Regulation of Neutrophil-Endothelial Cell Adhesion 
Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ETAreceptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.
doi:10.1251/bpo32
PMCID: PMC145555  PMID: 12734570
matrix metalloproteinases; endothelin-1
23.  Increased circulating 92 kDa matrix metalloproteinase (MMP-9) activity in exacerbations of asthma 
Thorax  2003;58(9):757-760.
Background: The 72 kDa matrix metalloproteinase 2 (MMP-2) and the 92 kDa matrix metalloproteinase 9 (MMP-9) are type IV collagenases implicated in various aspects of inflammation including accumulation of inflammatory cells, tissue injury, and development of remodelling. The role of these enzymes in the pathogenesis of asthma exacerbations is unknown.
Methods: Circulating levels of MMP-2 and MMP-9 proteins and the expression of their inhibitor, tissue inhibitor of metalloproteinase 1 (TIMP-1), were measured in 21 patients experiencing an asthma exacerbation and 21 age matched patients with stable asthma. Circulating gelatinolytic activity was compared during the asthma exacerbation and during subsequent convalescence by gelatin zymography in the same individuals. In addition, MMP-9 specific activity was quantified with a colorimetric assay which uses an artificial proenzyme containing a specific domain recognised by MMP-9 in the same paired samples.
Results: A significant increase in the circulating level of MMP-9 was seen in patients with an asthma exacerbation compared with patients with stable asthma (202.9 (22.0) v 107.7 (9.9) ng/ml, p=0.0003). There were no significant differences in the circulating levels of MMP-2 or TIMP-1. Gelatin zymography identified two major circulating gelatinolytic activities corresponding to MMP-2 and MMP-9, and showed that asthma exacerbations are characterised by markedly increased MMP-9 activity with no significant change in MMP-2 activity compared with the activities during convalescence in the same individuals. Direct measurement showed that MMP-9 specific activity is significantly increased during asthma exacerbations compared with subsequent convalescence (269.6 (31.7) v 170.4 (12.6) ng/ml, p=0.0099).
Conclusions: Asthma exacerbations are characterised by increased circulating MMP-9 activity. This increased activity may be related to exaggerated airway inflammation and airway remodelling.
doi:10.1136/thorax.58.9.757
PMCID: PMC1746799  PMID: 12947131
24.  Hepsin colocalizes with desmosomes and induces progression of ovarian cancer in a mouse model 
Hepsin is a serine protease that is widely expressed in different tissues and cell types, most prominently in the normal liver and kidney. Overexpression of hepsin has been associated with prostate cancers, ovarian cancers and renal cell carcinomas. The physiological functions of hepsin in normal tissues and tumors are poorly understood. To gain insight into its function in ovarian cancer, we analyzed the expression and subcellular localization of hepsin protein in ovarian cancer cell lines and tumors. We showed that the membrane-associated hepsin protein is present at desmosomal junctions, where it colocalizes with its putative proteolytic substrate hepatocyte growth factor. Consistent with the growing evidence that desmosomal junctions and their constituents play a role in cancer progression, we demonstrated that overexpression of hepsin promotes ovarian tumor growth in a mouse model. The ability of ectopic hepsin to induce tumor growth in mice is abrogated by the mutation of 3 critical residues in the catalytic domain, thus implicating the enzymatic activity of hepsin in promoting tumor progression.
doi:10.1002/ijc.23726
PMCID: PMC2653430  PMID: 18726901
desmosome; hepsin; ovarian cancer; serine protease
25.  Differential Effect of 17-β-Estradiol on Smooth Muscle Cell and Aortic Explant MMP2 
The Journal of surgical research  2008;155(1):48-53.
Objective
The present investigation tested the hypothesis that intrinsic gender-related differences exist in rat aortic smooth muscle cell MMP2.
Methods
This investigation comprised three sets of experiments. Experiment I: Adult male and female rat aortic smooth muscle cells (RASMCs) at passages 4–8 were stimulated in serum-free media for 48 hours with IL1β at doses encountered in human AAAs (2ng/mL). Messenger RNA was extracted from the RASMCs, and gene expression of MMP2 and tissue inhibitor of metalloproteinase2 (TIMP2),a major MMP2 inhibitor, was measured by real-time polymerase chain reaction. MMP2 protein levels in conditioned media were measured by Western Blotting, and MMP2 and TIMP2 activity quantified by standard and reverse gelatin zymography. Experiment II: Male and female RASMCs were incubated for 48 hrs in DMEM containing IL-1β and 17-β-estradiol at doses from 1×10−10 to 1×10−6 molar. MMP2 activity in the conditioned media was then determined. Experiment III: Male rats underwent sustained 17-β-estradiol exposure for 21 days using extended-release, subcutaneously implanted pellets prior to sacrifice and aortic explantation. Aortas from males, females, and estradiol-treated males were stimulated with IL1β for 48 hrs, and MMP2 activity in the conditioned media was determined.
Results
Experiment I: MMP2 gene expression was 3-fold higher in male compared to female IL1β stimulated RASMCs (P<0.0001). MMP2: TIMP2 gene expression ratio was 7.5 fold greater in male vs. female RASMCs. MMP2 protein levels were 3-fold higher (2.68 vs. 0.96 O.D./mg total protein, P=0.003) in male vs. female RASMCs. Gelatinolytic activity was more than 6-fold higher (15,010 vs. 2,472 O.D./mg total protein, P=0.002) in male vs. female RASMCs. Experiment II: MMP2 activity in male and female RASMCs was not altered by a wide range of 17-β-estradiol concentrations. Experiment III: When pre-treated with 17-β-estradiol, MMP2 activity in the media of male rat whole-aortic explants decreased two-fold ( P=0.002). This post-17-β-estradiol treatment male level was not different than baseline female aortic explant MMP2 levels.
Conclusions
MMP2 is higher in male RASMCs compared to female RASMCs. Exogenous 17-β-estradiol did not alter MMP2 activity in vitro, but in vivo 17-β-estradiol exposure greatly decreased male aortic MMP2 production to levels seen in the female aorta. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human AAA formation.
doi:10.1016/j.jss.2008.07.003
PMCID: PMC2913053  PMID: 19041098

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