Probes for INS

Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (HYBID/KIAA1199; encoded by CEMIP) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/locus of crossover in P1 (loxP) system and analyzed their phenotypes. Hybid-deficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4 weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction in the Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated that Hybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes. High-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondral ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.

Abstract: Objective: To investigate the potential involvement of secreted protein acidic and rich in cysteine (SPARC) in the progression of the breast tumor and to determine its association with outcome variables and matrix metalloproteinases (MMPs) expression in patients with breast carcinoma (BC). Methods: SPARC expression was examined in 8 pairs of BC tissues and surrounding normal tissues at mRNA and protein levels by qRT-PCR, RNAscope in situ hybridization (ISH), Western blotting, and immunohistochemistry techniques. Immunohistochemical staining of SPARC was done in 26 normal breasts, 76 ductal carcinoma in situ (DCIS), and 198 BC samples. In addition, immunohistochemical staining was performed for MMP-2 and MMP-9 in BC. Results: SPARC expression at mRNA and protein levels was significantly increased in BC tissues compared to the surrounding normal tissues (P < 0.05 and P < 0.01, respectively). RNAscope ISH and immunohistochemistry of SPARC confirmed an increase in SPARC expression in BC tissues compared with the normal tissues. Epithelial SPARC expression increased continuously from normal breast through DCIS to BC (P < 0.001). In patients with BC, high epithelial SPARC expression was associated with worse disease-free survival and overall survival (P = 0.002 and P = 0.048, respectively) and independently predicted worse disease-free survival (P = 0.002). Epithelial SPARC expression was significantly correlated with MMP-2 expression (P < 0.05). Conclusion: Up-regulation of SPARC contributes to breast tumor progression. SPARC expression may be a useful biomarker for the prognostic prediction in patients with BC. SPARC can control extracellular matrix degradation through up-regulation of MMP-2.

 Background: ASPM is a newly reported stem cell marker and plays important roles in mitosis, cell cycle and tumorigenesis. It links with poor clinical prognosis in various tumors. However, the clinical significance of ASPM in colonic adenocarcinoma (CA) has not been fully studied. The purpose of this study was to investigate if ASPM is correlated with the clinicopathological features of CA. Methods: Primary CA tissue, adenoma and the matched normal mucosa from 99 patients, were detected using immunohistochemical analysis by primary antibodies against ASPM. Meanwhile, 20 CAs and 20 liver metastatic cases were examined by RNA in situ hybridization (RNAscope). To assess the clinical relevance of ASPM, we analyzed the survival follow-up information. Results: ASPM was found only in single cells in the base of normal colon mucosal crypts. But the expression of ASPM was detected high in colonic adenomas (49.5%, 49/99), and significantly higher in CA (56.6%, 56/ 99, P<0.001). In CAs, ASPM expression was more intense in stage III and IV than II and I stage patients (P=0.03), and positively correlated with lymph node metastasis (P=0.03), but not with the age at diagnosis, gender and histological grade (P>0.05). We also analyzed the survival follow-up information, the data showed that ASPM-positive expression was correlated with a shorter disease-free survival (DFS) time, the average DFS time of patients with ASPM positive and negative expression was 62.79±2.32 months and 71.30±2.72 months, respectively, and there was no statistical significance between the two groups (P>0.05). The results of ASPM mRNA measurement by RNAscope revealed ASPM mRNA expression was higher in primary CA than that in metastatic liver CA (P<0.001). Conclusions: ASPM might play an important role in colonic carcinogenesis and be a potential marker in predicting prognosis of CA. 

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.

Abstract