MicroRNA 21a-5p overexpression impacts mediators of extracellular matrix formation in uterine leiomyoma.

BACKGROUND: MicroRNAs (MiR) may promote fibroid development via altered expression of genes involved in cell proliferation and ECM formation, and evidence supports aberrant expression of MicroRNA (MiR) 21a-5p in fibroids. The purpose of this study was to investigate the functional significance of MiR 21a-5p overexpression in the pathobiology of leiomyomata (fibroids). METHODS: A basic science experimental design using immortalized fibroid and myometrial cell lines derived from patient-matched specimens was used. Stable overexpression of MiR-21a-5p in an immortalized fibroid and patient matched myometrial cell line was achieved through lentiviral vector infection. Main outcome measures were MiR-21-5p overexpression, target gene and protein expression, collagen (COL1A1) production, cell proliferation, cell migration, and cell cycle stages of fibroid and myometrial immortalized cell lines. RESULTS: MiR-21a-5p was overexpressed to similar levels in fibroid and myometrial cell lines after lentiviral infection. Increased expression of miR-21 resulted in increased gene and protein expression of TGF-ß3 in both fibroid and myometrial cells. Changes in expression of the ECM genes Fibronectin, Collagen 1A1, CTGF, Versican and DPT were seen in both fibroid and myometrial cells. Changes were also seen in Matrix Metalloproteinase (MMP) related genes including MMP 2, MMP 9, MMP 11 and Serpine 1 in both fibroid and myometrial cells. MiR-21 upregulation resulted in increased proliferation and migration in fibroid cells compared to myometrial cells. CONCLUSIONS: MiR-21a-5p overexpression results in changes in the expression of ECM mediators in both fibroid and myometrial cells, and increased cell proliferation in fibroid cells. These finding suggest a potential functional role of MiR-21a-5p in the development of uterine fibroids and warrant further investigation.

The COOH-terminal domain of huntingtin interacts with RhoGEF kalirin and modulates cell survival.

Human huntingtin (Htt) contains 3144 amino acids and has an expanded polyglutamine region near the NH2-terminus in patients with Huntington's disease. While numerous binding partners have been identified to NH2-terminal Htt, fewer proteins are known to interact with C-terminal domains of Htt. Here we report that kalirin, a Rac1 activator, is a binding partner to C-terminal Htt. Kalirin and Htt co-precipitated from mouse brain endosomes and co-localized at puncta in NRK and immortalized striatal cells and primary cortical neurons. We mapped the interaction domains to kalirin674-1272 and Htt2568-3144 and determined that the interaction between kalirin and Htt was independent of HAP1, a known interactor for Htt and kalirin. Kalirin precipitated with mutant Htt was more abundant than with wild-type Htt and had a reduced capacity to activate Rac1 when mutant Htt was present. Expression of Htt2568-3144 caused cytotoxicity, partially rescued by co-expressing kalirin674-1272 but not other regions of kalirin. Our study suggests that the interaction of kalirin with the C-terminal region of Htt influences the function of kalirin and modulates the cytotoxicity induced by C-terminal Htt.

Glucose transporter 3 is a rab11-dependent trafficking cargo and its transport to the cell surface is reduced in neurons of CAG140 Huntington's disease mice.

Huntington's disease (HD) disturbs glucose metabolism in the brain by poorly understood mechanisms. HD neurons have defective glucose uptake, which is attenuated upon enhancing rab11 activity. Rab11 regulates numerous receptors and transporters trafficking onto cell surfaces; its diminished activity in HD cells affects the recycling of transferrin receptor and neuronal glutamate/cysteine transporter EAAC1. Glucose transporter 3 (Glut3) handles most glucose uptake in neurons. Here we investigated rab11 involvement in Glut3 trafficking. Glut3 was localized to rab11 positive puncta in primary neurons and immortalized striatal cells by immunofluorescence labeling and detected in rab11-enriched endosomes immuno-isolated from mouse brain by Western blot. Expression of dominant active and negative rab11 mutants in clonal striatal cells altered the levels of cell surface Glut3 suggesting a regulation by rab11. About 4% of total Glut3 occurred at the cell surface of primary WT neurons. HD(140Q/140Q) neurons had significantly less cell surface Glut3 than did WT neurons. Western blot analysis revealed comparable levels of Glut3 in the striatum and cortex of WT and HD(140Q/140Q) mice. However, brain slices immunolabeled with an antibody recognizing an extracellular epitope to Glut3 showed reduced surface expression of Glut3 in the striatum and cortex of HD(140Q/140Q) mice compared to that of WT mice. Surface labeling of GABAα1 receptor, which is not dependent on rab11, was not different between WT and HD(140Q/140Q) mouse brain slices. These data define Glut3 to be a rab11-dependent trafficking cargo and suggest that impaired Glut3 trafficking arising from rab11 dysfunction underlies the glucose hypometabolism observed in HD.