Kindlin-2 deficiency induces fatal intestinal obstruction in mice.

Rationale: Smooth muscle-motility disorders are mainly characterized by impaired contractility and functional intestinal obstruction. Some of these cases are caused by genetic mutations of smooth muscle genes ACTA2, ACTG2, MYH11, MYLK and LMOD1. Still the etiology is complex and multifactorial and the underlying pathology is poorly understood. Integrin interaction protein Kindlin-2 is widely expressed in striated and smooth muscle cells (SMC). However, the function of Kindlin-2 in the smooth muscle remains elusive. Methods: We generated two mouse models using different cre promoter transgenic mice, Kindlin-2fl/fl SM22α-cre+ (cKO mice) and Kindlin-2fl/fl; MYH-cre+ (iKO mice). Embryos and adult tissues were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay. We investigated ultrastructure changes of mouse smooth muscle using transmission electron microscopy (TEM) and measured smooth muscle contractile force in mounting aortic and intestinal rings using the multiwire myograph system (DMT 620M). In addition, cell traction force microscopy (CTFM) was applied to observe the functional change of primary SMC after Kindlin-2 depletion by RNAi. Results: Depletion of Kindlin-2 encoding gene Fermt2 in embryonic smooth muscles leads to apoptosis, downregulates the key components of SMC, impairs smooth muscle development, and finally causes embryonic death at E14.5. Tamoxifen-induced Kindlin-2-specific knockout in adult mouse smooth muscle showed decreased blood pressure, intestinal hypoperistalsis, and eventually died of intestinal obstruction. Kindlin-2 depletion also leads to downregulated Myh11, α-SMA, and CNN, shortened myofilament, broken myofibrils, and impaired contractility of the smooth muscles in iKO mice. Mechanistically, loss of Kindlin-2 decreases Ca2+ influx in primary vascular smooth muscle cells (PVSMC) by downregulating the expression of calcium-binding protein S100A14 and STIM1. Conclusion: We demonstrated that Kindlin-2 is essential for maintaining the normal structure and function of smooth muscles. Loss of Kindlin-2 impairs smooth muscle formation during embryonic development by inducing apoptosis and jeopardizes the contraction of adult smooth muscle by blocking Ca2+ influx that leads to intestinal obstruction. Mice with Kindlin-2 depletion in adult smooth muscle could be a potent animal model of intestinal obstruction for disease research, drug treatment and prognosis.

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice.

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and ß-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

Unconventional myosin VIIA promotes melanoma progression.

Unconventional myosin VIIA (Myo7a) is an actin-based motor molecule that normally functions in the cochlear hair cells of the inner ear. Mutations of MYO7A/Myo7a have been implicated in inherited deafness in both humans and mice. However, there is limited information about the functions of Myo7a outside of the specialized cells of the ears. Herein, we report a previously unidentified function of Myo7a by demonstrating that it plays an important role in melanoma progression. We found that silencing Myo7a by means of RNAi inhibited melanoma cell growth through upregulation of cell cycle regulator p21 (also known as CDKN1A) and suppressed melanoma cell migration and invasion through downregulation of RhoGDI2 (also known as ARHGDIB) and MMP9. Furthermore, Myo7a depletion suppressed melanoma cell metastases to the lung, kidney and bone in mice. In contrast, overexpression of Myo7a promoted melanoma xenograft growth and lung metastasis. Importantly, Myo7a levels are remarkably elevated in human melanoma patients. Collectively, we demonstrated for the first time that Myo7a is able to function in non-specialized cells, a finding that reveals the complicated disease-related roles of Myo7a, especially in melanomas.

High expression of muscarinic acetylcholine receptor 3 predicts poor prognosis in patients with pancreatic ductal adenocarcinoma.

AIMS: Recent studies showed that muscarinic acetylcholine receptor 3 (M3), as a muscarinic acetylcholine receptor family member that plays an important role in normal physiological function, is engaged in cancer progression. However, the role of M3 in pancreatic ductal adenocarcinoma (PDAC) is not known. The aim of this study is to investigate the expression and prognostic value of M3 in patients with PDAC. MATERIALS AND METHODS: The localization and expression of M3 in PDAC were examined by immunohistochemistry. VAChT was employed to detect parasympathetic nerve fibers in the corresponding M3 PDAC tissues. The correlation between M3 expression and patients' survival was assessed by Kaplan-Meier analysis. RESULTS: M3 was discovered predominantly localized in the cell cytoplasm and expressed in all specimens of PDAC patients. Significant correlation was noted between increased M3 intensity and high grade of PDAC (P<0.01), more lymph node metastasis (P<0.01) as well as shorter patient overall survival (P<0.01). Morphologically, cells with high M3 expression were more frequently located at the invasive tumor front/tumor budding cells, metastatic lymph nodes and parasympathetic nerve fibers. CONCLUSION: High expression of M3 is a prognostic marker for PDAC.

[Study of clinical value of dual-phase imaging using single photon emission computed tomography with coincidence detection for differentiating benign from malignant lesions].

OBJECTIVE: To investigate the clinical value of dual-phase imaging of 18F-fluoro-2-deoxy-D-glucose (18F-FDG) using single photon emission computed tomography with coincidence detection (SPECT with coincidence) for differentiating benign and malignant lesions. METHODS: Thirty patients with unknown lesions from June 2003 to March 2004 in our hospital were enrolled in this study. All patients underwent dual-phase imaging of 18F-FDG SPECT with coincidence. The early images were obtained 60 min post injection. And the patients were divided into 2 groups, one receiving delayed scans at 210 min after 18F-FDG injection and the other at 270 min. Lesion status was determined by pathologic findings or by clinical follow-up. RESULTS: Forty-eight lesions (30 malignant and 18 benign lesions verified by pathologic findings or clinical follow-up) of 30 patients were detected by dual-phase imaging, in which there were 29 true positive results, 15 true negative, 3 false positive and 1 false negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of dual-phase imaging were 96.7%, 83.3%, 91.6%, 90.6%, and 93.8%, respectively. Those of early imaging and CT were 93.3%, 72.2%, 85.4%, 84.8%, 86.7% and 76.9%, 57.1%, 70.0%, 76.9%, 57.1%, respectively. The T/NT of all malignant lesions (except 1 false negative case) in delayed imaging were significantly increased (t=-3.071, P<0.01). However, there were no statistical differences in the T/NT of benign lesions (t=0.398, P=0.695) between early and delayed imagings. CONCLUSION: Dual-phase imaging of FDG SPECT with coincidence provides benefit for differentiating benign and malignant lesions.