Demethylation of TIMP2 and TIMP3 Inhibits Cell Proliferation, Migration, and Invasion in Pituitary Adenomas.

OBJECTIVE: Tissue inhibitors of matrix metalloproteinases (TIMPs) are prognostic markers in cancers. However, the role of TIMPs in DNA methylation during invasive pituitary adenoma (PA) remains unclear. The purpose of this study was to assess the effects of TIMP2 and TIMP3 promoter demethylation on the proliferation, migration, and invasion of invasive PA cells. METHODS: Methylation-specific polymerase chain reaction (PCR), quantitative PCR, and western blots were used to analyze the promoter methylation and expression of TIMP1-3. Cell counting kit-8 (CCK-8), wound healing, and transwell assays were carried out to determine the effects of TIMP2 and TIMP3 demethylation. RESULTS: TIMP1-3 showed downregulated expression in invasive PA tissues and cell lines (p < 0.05). The low expression of TIMP1-3 was due to promoter methylation of these genes (p < 0.05). The results showed that downregulation of TIMP2 and TIMP3 can promote cell proliferation, migration, and invasion (p < 0.05), whereas overexpression of TIMP2 and TIMP3 can inhibit cell proliferation, migration, and invasion (p < 0.05). After treatment with 5-azacytidine (5-AzaC), the cell activity decreased, the proliferation rate decreased, and the invasion ability weakened (p < 0.05). Treatment with 5-AzaC increased TIMP2 and TIMP3 expression and decreased DNA (cytosine-5-)-methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b expression (p < 0.05). CONCLUSIONS: We showed that DNA methylation causes the silencing of TIMP2 and TIMP3 in invasive PA, it can also lead to malignant cell proliferation and cause pathological changes, whereas the use of 5-AzaC can inhibit the methylation process and can inhibit cell proliferation. Our results provide a novel method for clinical diagnosis and prevention of invasive PA.

Tissue Inhibitor of Metalloproteinase 3: Unravelling Its Biological Function and Significance in Oncology.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is vital in regulating several biological processes. TIMP3 exerts antitumour effects via matrix metalloproteinase (MMP)-dependent and MMP-independent pathways. Due to promoter methylation and miRNA binding, TIMP3 expression has been observed to decrease in various cancers. Consequently, the migration and invasion of cancer cells increases. Conflicting results have reported that expression levels of TIMP3 in primary and advanced cancers are higher than those in healthy tissues. Therefore, the role of TIMP3 in cancer biology and progression needs to be elucidated. This review provides an overview of TIMP3, from its biological function to its effects on various cancers. Moreover, gynaecological cancers are discussed in detail. TIMP3 has been associated with cervical adenocarcinoma as well as cancer development in serous ovarian cancer and breast cancer metastasis. However, the relationship between TIMP3 and endometrial cancers remains unclear. TIMP3 may be a useful biomarker for gynaecological cancers and is a potential target for future cancer therapy.

Abnormal stress promotes intervertebral disc degeneration through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2-mediated m6A modification in abnormal stress-induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Sublytic C5b-9 induces TIMP3 expression by glomerular mesangial cells via TRAF6-dependent KLF5 K63-linked ubiquitination in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Generation of a TIMP3 knockout stem cell line via CRISPR/Cas9 system.

The tissue inhibitors of metalloproteinases 3 (TIMP3) play an essential role in the tumorigenesis of human pancreatic endocrine tumors and Sorsby fundus dystrophy. To further investigate the significance of TIMP3 in disease, we used CRISPR/Cas9 to create a TIMP3 knock out human embryonic stem cell line (WAe009-A-89) that can differentiate into any desired cell type. Our results show that the WAe009-A-89 cell line retains the typical colony form and normal karyotype of stem cells. The cells strongly expressed pluripotency markers and could differentiate into tissues of all three germ layers in vivo. This cell line allowed exploring the role of the TIMP3 gene in related diseases.

Clinicopathological features of rhabdomyosarcoma with novel FET::TFCP2 and TIMP3::ALK fusion: report of two cases and literature review.

AIMS: The aim of this study was to evaluate the clinicopathological features, immunophenotype, differential diagnosis, molecular genetic features and prognosis of spindle cell rhabdomyosarcoma with TFCP2 rearrangement. METHODS: Two cases of spindle cell rhabdomyosarcoma with FET::TFCP2 gene fusion were included in this study. Samples were collected and evaluated through histological observation, immunohistochemistry, fluorescence in-situ hybridisation and high-throughput gene sequencing and previous findings. RESULTS: The tumour tissues mainly comprised spindle cells and epithelioid cells, which expressed striated muscle markers, and exhibited high expression levels of CK and ALK protein markers. Molecular detection showed that the FET::TFCP2 gene was fused. A rare case with TIMP3::ALK and FUS::TFCP2 double-fusion was observed in this study. CONCLUSIONS: A case with double fusion of ALK and TFCP2 was reported in rhabdomyosarcoma for the first time in this study, which provides information on the molecular characteristic of the tumour. Spindle cell rhabdomyosarcoma with FET::TFCP2 fusion is characterised by histological, immunohistochemical and genetic changes. The tumour is aggressive, with poor prognosis and poor response to radiotherapy and chemotherapy. The efficacy of targeted therapy for ALK should be explored through more clinical studies.

Hypertension Promotes the Proliferation and Migration of ccRCC Cells by Downregulation of TIMP3 in Tumor Endothelial Cells through the miR-21-5p/TGFBR2/P38/EGR1 Axis.

Recent studies have demonstrated that hypertension correlates with tumorigenesis and prognosis of clear-cell renal cell carcinoma (ccRCC); however, the underlying molecular mechanisms remain unclear. By analyzing bulk and single-cell RNA sequencing data and experimental examining of surgical excised ccRCC samples, we found that tissue inhibitors of metalloproteinases 3 (TIMP3), a pivotal paracrine factor in suppressing tumor progression, was significantly reduced in the tumor endothelial cells of patients with hypertensive ccRCC. Besides, in tumor xenograft of NCG mouse model, compared with saline normotensive group the expression of TIMP3 was significantly decreased in the angiotensin II-induced hypertension group. Treating human umbilical vein endothelial cells (HUVEC) with the plasma of patients with hypertensive ccRCC and miR-21-5p, elevated in the plasma of patients with hypertensive ccRCC, reduced the expression of TIMP3 compared with normotensive and control littermates. We also found that the inhibition of TIMP3 expression by miR-21-5p was not through directly targeting at 3'UTR of TIMP3 but through suppressing the expression of TGFß receptor 2 (TGFBR2). In addition, the knockout of TGFBR2 reduced TIMP3 expression in HUVECs through P38/EGR1 (early growth response protein 1) signaling axis. Moreover, via coculture of ccRCC cell lines with HUVECs and mouse tumor xenograft model, we discovered that the TIMP3 could suppress the proliferation and migration of ccRCC. IMPLICATIONS: Overall, our findings shed new light on the role of hypertension in promoting the progression of ccRCC and provide a potential therapeutic target for patients with ccRCC with hypertension.

TIMP3 overexpression in myeloid lineage alleviates pancreatic damage and confers resistance to the development of type 1 diabetes in the MLDS -induced model.

Introduction: Type 1 diabetes mellitus (T1DM) development involves a complex interplay of genetic, environmental, and immunological factors. By modulating the activity of proteases and receptors, the protein tissue inhibitor of metalloproteinase 3 (TIMP3) plays a role in limiting the expression and function of pro-inflammatory cytokines, which have been implicated in the advancement of T1DM. This study was aimed at examining the effect of TIMP3 overexpression in myeloid cells on the development of T1DM. Methods and results: Twelve weeks after multiple low doses of streptozotocin (MLDS) treatment, diabetic mice overexpressing TIMP3 specifically in myeloid cells under the CD68 promoter (MacT3 mice) showed improved insulin secretion, islet morphology and vascularization, antioxidant defense system, and regulatory factors of mitochondrial biosynthesis and function. To get mechanistic insights into the origin of this protection, the severity of insulitis and inflammatory parameters were evaluated in pancreatic tissues 11 days after MLSD treatment, showing significantly reduced insulitis and levels of the pro-inflammatory cytokine tumor necrosis factor-α, interleukin -1ß, and interferon -γ in MacT3 mice. Discussion: The results indicate that TIMP3 is involved in maintaining islet architecture and functions, at least in part, through modulation of pro-inflammatory cytokine production associated with insulitis and may represent a novel therapeutic strategy for T1DM.

TIMP3 Gene Polymorphisms of -1296 T > C and -915 A > G Increase the Susceptibility to Arsenic-Induced Skin Cancer: A Cohort Study and In Silico Analysis of Mutation Impacts

Long-term exposure to arsenic may induce several human cancers, including non-melanoma skin cancer. The tissue inhibitor of metalloproteinase (TIMP)-3, encoded by the TIMP3 gene, may inhibit tumor growth, invasion, and metastasis of several cancer types. In this study, we aimed to investigate effects of the TIMP3 -1296 T > C (rs9619311) and -915 A > G (rs2234921) single-nucleotide polymorphisms (SNPs) on skin cancer risk in an arsenic-exposed population, and to evaluate the influence of allele-specific changes by an in silico analysis. In total, 1078 study participants were followed up for a median of 15 years for newly diagnosed skin cancer. New cases were identified through linkage to the National Cancer Registry of Taiwan. A Cox regression analysis was used to evaluate the effects of TIMP3 variants. Transcription factor (TF) profiling of binding sites of allele-specific changes in SNPs was conducted using the JASPAR scan tool. We observed borderline associations between TIMP3 genotypes and skin cancer risk. However, when combined with high arsenic exposure levels, the rs9619311 C allele, rs2234921 G allele, or C-G haplotype groups exhibited a greater risk of developing skin cancer compared to the respective common homozygous genotype group. The in silico analysis revealed several TF motifs located at or flanking the two SNP sites. We validated that the C allele of rs9619311 attenuated the binding affinity of BACH2, MEIS2, NFE2L2, and PBX2 to the TIMP3 promoter, and that the G allele of rs2234921 reduced the affinity of E2F8 and RUNX1 to bind to the promoter. Our findings suggest significant modifications of the effect of the association between arsenic exposure and skin cancer risk by the TIMP3 rs9619311 and rs2234921 variants. The predicted TFs and their differential binding affinities to the TIMP3 promoter provide insights into how TIMP3 interacts with arsenic through TFs in skin cancer formation.

Deglycosylation Increases the Aggregation and Angiogenic Properties of Mutant Tissue Inhibitor of Metalloproteinase 3 Protein: Implications for Sorsby Fundus Dystrophy.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular disorder caused by mutations in tissue Inhibitor of the metalloproteinase-3 (TIMP3) gene with the onset of symptoms including choroidal neovascularization as early as the second decade of life. We have previously reported that wild-type TIMP3 is an endogenous angiogenesis inhibitor that inhibits Vascular Endothelial Growth Factor (VEGF)-mediated signaling in endothelial cells. In contrast, SFD-related S179C-TIMP3 when expressed in endothelial cells, does not have angiogenesis-inhibitory properties. To evaluate if this is a common feature of TIMP3 mutants associated with SFD, we examined and compared endothelial cells expressing S179C, Y191C and S204C TIMP3 mutants for their angiogenesis-inhibitory function. Western blot analysis, zymography and reverse zymography and migration assays were utilized to evaluate TIMP3 protein, Matrix Metalloproteinase (MMP) and MMP inhibitory activity, VEGF signaling and in vitro migration in endothelial cells expressing (VEGF receptor-2 (VEGFR-2) and wild-type TIMP3 or mutant-TIMP3. We demonstrate that mutant S179C, Y191C- and S204C-TIMP3 all show increased glycosylation and multimerization/aggregation of the TIMP3 protein. In addition, endothelial cells expressing TIMP3 mutations show increased angiogenic activities and elevated VEGFR-2. Removal of N-glycosylation by mutation of Asn184, the only potential N-glycosylation site in mutant TIMP3, resulted in increased aggregation of TIMP3, further upregulation of VEGFR-2, VEGF-induced phosphorylation of VEGFR2 and VEGF-mediated migration concomitant with reduced MMP inhibitory activity. These results suggest that even though mutant TIMP3 proteins are more glycosylated, post-translational deglycosylation may play a critical role in the aggregation of mutant TIMP3 and contribute to the pathogenesis of SFD. The identification of factors that might contribute to changes in the glycome of patients with SFD will be useful. Future studies will evaluate whether variations in the glycosylation of mutant TIMP3 proteins are contributing to the severity of the disease.

Sorsby fundus dystrophy (SFD): A narrative review.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disorder with complete penetrance affecting the macula. This is caused by a mutation in the TIMP-3. This objective narrative review aims to provide an overview of the pathophysiology, current treatment modalities, and future perspectives. A literature search was performed using "PubMed," "Web of Science," "Scopus," "ScienceDirect," "Google Scholar," "medRxiv," and "bioRxiv." The molecular mechanisms underlying SFD are not completely understood. Novel advancements in cell culture techniques, including induced pluripotent stem cells, may enable more reliable modeling of SFD. These cell culture techniques aim to shed more light on the pathophysiology of SFD, and hopefully, this may lead to the future development of treatment strategies for SFD. Currently, no gene therapy is available. The main treatment is the use of anti-vascular endothelial growth factors (anti-VEGF) to treat secondary choroidal neovascular membrane (CNV), which is a major complication observed in this condition. If CNV is detected and treated promptly, patients with SFD have a good chance of maintaining a functional central vision. Other treatment modalities have been tried but have shown limited benefit, and therefore, have not managed to be more widely accepted. In summary, although there is no definitive cure yet, the use of anti-VEGF treatment for secondary CNV has provided the opportunity to maintain functional vision in individuals with SFD, provided CNV is detected and treated early.

MicroRNA-613 Enhances Nasopharyngeal Carcinoma Cell Radiosensitivity via the DNA Methyltransferase 3B/Tissue Inhibitor of Matrix Metalloproteinase-3/Signal Transducer and Activator of Transcription-1/Forkhead Box O-1 Axis.

Nasopharyngeal carcinoma (NPC) is a common malignancy of the nasopharynx, and radioresistant represents the main obstacle in NPC treatment. Malignant transformation of normal cells is driven by genetic and epigenetic changes, which are primarily manifested as changes in miRNA levels and DNA methylation status. microRNA (miR)-613 plays an inhibitory role in several types of cancer. Herein, the current study sought to explore the roles of miR-613 in NPC cell radiosensitivity. miR-613 expression patterns in NPC tissues were detected, and its correlation with clinical indexes was analyzed. NP-69 and C666-1 cell lines were selected for cellular experimentation. Radioresistant cell line C666-1R was obtained by fractionated radiation. Cell viability, survival fraction, and apoptosis were detected by CCK-8, colony formation assay, and flow cytometry. The binding relation between miR-613 and DNMT3B was verified by dual-luciferase and RIP assays. miR-613 was lowly expressed in NPC tissues and cells, with lower expression levels in C666-1R than C666-1, and further correlated with lymph node metastasis, tumor size, and tumor metastasis. miR-613 overexpression reduced C666-1R cell viability and survival fraction and increased apoptosis, while C666-1 cells with silencing miR-613 presented the opposite trends. miR-613 targeted DNMT3B. miR-613 and DNMT3B overexpression led to enhanced C666-1R cell viability and survival fraction and decreased apoptosis. miR-613 reduced TIMP3 methylation and elevated TIMP3 protein level by inhibiting DNMT3B. miR-613 enhanced NPC radiosensitivity by inhibiting the DNMT3B/TIMP3/STAT1/FOXO1 pathway. Collectively, miR-613 inhibited DNMT3B, reduced TIMP3 methylation, and increased TIMP3 protein level, thus inhibiting the STAT1/FOXO1 pathway and enhancing the radiosensitivity of NPC cells.

Exosomal miR-452-5p Induce M2 Macrophage Polarization to Accelerate Hepatocellular Carcinoma Progression by Targeting TIMP3.

Background: Hepatocellular carcinoma (HCC) cell-derived exosomes have shown effects on inducing M2 macrophage polarization and promoting HCC progression. MiR-452-5p was reported by recent studies to promote malignancy progression as an exosomal microRNA that secreted by HCC cells, of which the underlying mechanism remains unclear. Here, we further explored how miR-452-5p functions in HCC. Methods: MiR-452-5p expressions in HCC cells was examined by in situ hybridization. Next, HCC cell lines were transfected with the mimics or the inhibitor of miR-452-5p. Transfected cells' biological behavior were analyzed by CCK-8, flow cytometry, and Transwell assay. Then, exosomes were purified from miR-452-5p inhibited or overexpressed HCC cells and cocultured with macrophages to examine the role of miR-452-5p in macrophage polarization. To examine the role of exosomal miR-452-5p on macrophage polarization and tumor growth. We also performed the dual-luciferase assay to explore the targeting relationship between miR-452-5p and TIMP3. Results: The upregulation of miR-452-5p was identified in HCC. The effects of HCC cell-derived exosomes on accelerating HCC migration and invasion and inducing M2 macrophage polarization were confirmed, which were further enhanced after overexpressing miR-452-5p but neutralized after silencing miR-452-5p. In addition, in vivo experiments demonstrated the effect of miR-452-5p on accelerating HCC growth and metastasis. Also, we identified that TIMP3 overexpression inhibited the promoted cell invasion and migration by HCC cell-derived exosomes. Conclusion: Exosomal miR-452-5p secreted from HCC cells could induce polarization of M2 macrophage and therefore stimulating HCC progression by targeting TIMP3. Thus, miR-452-5p might be a potential biomarker for HCC prognosis.

TIMP3::ALK fusions characterize a distinctive myxoid fibroblastic tumor of the vocal cords: a report of 7 cases.

We report 7 cases of an indolent, variably myxoid tumor of the vocal cords, characterized by overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles, delicate curvilinear vessels, and sparse inflammatory infiltrate. Six patients were male, aged 15 to 65 years, and 1 patient was a 54-year-old female. All tumors were located in the superficial portion of the vocal cord. One patient suffered a recurrence that was completely resected; all patients with available follow-up data currently have no evidence of disease. The tumors contained alternating areas with myxoid stroma and more compacted regions with tumor cells organized in short fascicles, interwoven with delicate curvilinear vasculature. Overt cellular atypia with large cells containing intranuclear and intracytoplasmic vacuoles or resembling ganglion cells was present in all cases but mitoses and necrosis were absent. ALK immunostaining was positive in all cases, while most tumors were negative for smooth muscle actin. Targeted RNA-sequencing revealed an identical TIMP3::ALK fusion with exon 1 of TIMP3 gene being fused with exon 12 of ALK gene in all analyzable cases. For various reasons discussed, it remains unclear whether this tumor represents a mere subtype of IMT or a separate entity. Nevertheless, it is a morphologically distinct and diagnostically challenging lesion that needs to be recognized by surgical pathologists in order to prevent overdiagnosis in this clinically very delicate area.

Effect of tissue inhibitor of metalloproteinases-3 genetics polymorphism on clinicopathological characteristics of uterine cervical cancer patients in Taiwan.

Single nucleotide polymorphisms (SNPs) of tissue inhibitor of metalloproteinases-3 (TIMP-3) have been revealed to be related to various cancers. To date, no study explores the relationships between TIMP-3 polymorphisms and uterine cervical cancer. The purposes of this research were to investigate the associations among genetic variants of TIMP-3 and development and clinicopathological factors of uterine cervical cancer, and patient 5 years survival in Taiwanese women. The study included 123 patients with invasive cancer and 97 with precancerous lesions of uterine cervix, and 300 control women. TIMP-3 polymorphisms rs9619311, rs9862 and rs11547635 were checked and their genotypic distributions were determined by real-time polymerase chain reaction. It showed that women with genotypes CT/TT in rs9862 were found to display a higher risk of developing cervical cancer with moderate and poor cell differentiation. Moreover, it revealed that cervical cancer patients carrying genotypes CC in rs9619311 exhibited a poorer 5 years survival, as compared to those with TT/TC in Taiwanese women, using univariate analysis. In addition, pelvic lymph node metastasis was determined to independently predict 5 years survival in cervical cancer patients using multivariate analysis. Conclusively, TIMP-3 SNPs polymorphisms rs9619311 are related to cervical patient survival in Taiwanese women.

MicroRNA-574 Impacts Granulosa Cell Estradiol Production via Targeting TIMP3 and ERK1/2 Signaling Pathway.

Estradiol represents a key steroid ovarian hormone that not only plays a vital role in ovarian follicular development but also is associated with many other reproductive functions. Our primary study revealed that miR-574 expression decreased in porcine granulosa cells during development from small to large follicles, and the increase of ERK1/2 phosphorylation accompanies this change. Since it has been well established that the ERK1/2 activity is tightly associated with granulosa cell functions, including ovarian hormone production, we thus further investigate if the miRNA is involved in the regulation of estradiol production in granulosa cells. We found that overexpression of miR-574 decreased phosphorylated ERK1/2 without affecting the level of ERK1/2 protein, and on the other hand, the inhibition of miR-574 increased phosphorylated ERK1/2 level (P<0.05); meanwhile, overexpression of miR-574 increased estradiol production but knockdown of miR-574 decreased estradiol level in granulosa cells. To further identify the potential mechanism involved in the miR-574 regulatory effect, in silico screening was performed and revealed a potential binding site on the 3'UTR region of the tissue inhibitor of metalloproteinase 3 (TIMP3). Our gain-, loss- of function experiments, and luciferase reporter assay confirmed that TIMP3 is indeed the target of miR-574 in granulosa cell. Furthermore, the siRNA TIMP3 knockdown resulted in decreased phosphorylated ERK1/2, and an increase in estradiol production. In contrast, the addition of recombinant TIMP3 increased phosphorylated ERK1/2 level and decreased estradiol production. In summary, our results suggest that the miR-574-TIMP3-pERK1/2 cascade may be one of the pathways by which microRNAs regulate granulosa cell estradiol production.

Paracrine effect of the stromal vascular fraction containing M2 macrophages on human chondrocytes through the Smad2/3 signaling pathway.

The adipose-derived stromal vascular fraction (SVF) is composed of a heterogeneous mix of adipose-derived stem cells (ADSCs), macrophages, pericytes, fibroblasts, blood, and other cells. Previous studies have found that the paracrine effects of SVF cells may be therapeutic, but their role in osteoarthritis treatment remains unclear. This study aimed to investigate the therapeutic effect of SVF cells on chondrocytes. Chondrocytes were seeded on culture plates alone (control) or cocultured with SVF or ADSCs on cell culture inserts. After 48 h of coculture, chondrocyte collagen II, tissue inhibitors of metalloproteinases-3 (TIMP-3), and matrix metalloproteinases-13 (MMP-13) messenger RNA (mRNA) expression levels were evaluated using reverse-transcription polymerase chain reaction, and the transforming growth factor-ß (TGF-ß) levels in the supernatant were measured using ELISA. Immunohistochemical staining and flow cytometry were used to evaluate the macrophages in the SVF. These macrophages were characterized according to phenotype using the F4/80, CD86, and CD163 markers. To determine whether the Smad2/3 signaling pathways were involved, the chondrocytes were pre-treated with a Smad2/3 phosphorylation inhibitor and stimulated with the SVF, and then Smad2/3 phosphorylation levels were analyzed using western blot. The mRNA expression levels of various paracrine factors and chondrocyte pellet size were also assessed. Collagen II and TIMP-3 expression were higher in the SVF group than in the ADSC group and controls, while MMP-13 expression was the highest in the ADSC group and the lowest in the controls. TGF-ß levels in the SVF group were also elevated. Immunohistochemical staining and flow cytometry revealed that the macrophages in the SVF were of the anti-inflammatory phenotype. Western blot analysis showed that the SVF increased Smad2/3 phosphorylation, while Smad2/3 inhibitors decreased phosphorylation. Smad2/3 inhibitors also reduced the expression of various other paracrine factors and decreased chondrocyte pellet size. These findings suggested that the paracrine effect of heterogeneous cells, such as anti-inflammatory macrophages, in the SVF partly supports chondrocyte regeneration through TGF-ß-induced Smad2/3 phosphorylation.

Trehalose Protects Keratinocytes against Ultraviolet B Radiation by Activating Autophagy via Regulating TIMP3 and ATG9A.

Trehalose, a natural disaccharide, is synthesized by many organisms when cells are exposed to stressful stimuli. On the basis of its ability to modulate autophagy, trehalose has been considered an innovative drug for ameliorating many diseases, but its molecular mechanism is not well described. Previous findings demonstrated that trehalose plays a photoprotective role against ultraviolet (UV) B-induced damage through autophagy induction in keratinocytes. In this study, coimmunoprecipitation, label-free quantitative proteomic and parallel reaction monitoring, and western blot analysis demonstrated that trehalose promotes the interaction between tissue inhibitor of metalloproteinase (TIMP) 3 and Beclin1. Western blot and immunofluorescence staining analysis suggested that trehalose increases ATG9A localization in lysosomes and decreases its localization in the endoplasmic reticulum. Furthermore, in the presence or absence of UVB radiation, we evaluated the influence of TIMP3 and ATG9A small interfering RNA (siRNA) on the effect of trehalose on autophagy, cell death, migration, or interleukin-8 expression in keratinocytes, including HaCaT, A431, and human epidermal keratinocytes. The results revealed that in HaCaT cells, TIMP3 and ATG9A siRNA resulted in attenuation of trehalose-induced autophagy and inhibited cell death. In A431 cells, TIMP3 and ATG9A siRNA led to attenuation of trehalose-induced autophagy and cell death and inhibited migration. In human epidermal keratinocytes, trehalose-induced autophagy and inhibition of the interleukin-8 expression were blocked by ATG9A but not TIMP3 siRNA. In addition, the results of quantitative real-time PCR and immunohistochemistry analysis demonstrated the abnormal expression of TIMP3 and ATG9A in actinic keratosis and cutaneous squamous cell carcinoma skin tissues. These findings suggest the protective effects of trehalose in normal keratinocytes and its inhibitory effects on cancerous keratinocytes, possibly mediated by activation of autophagy and regulation of TIMP3 and ATG9A, providing the mechanistic basis for the potential use of trehalose in the prevention or treatment of UVB-induced skin diseases.