The incidence of diabetes is escalating worldwide and consequently, this has become a major healthcare problem. Moreover, both type 1 and type 2 diabetes are associated with significantly accelerated rates of microvascular complications including retinopathy, nephropathy and neuropathy, as well as macrovascular complications such as atherosclerotic cardiovascular and hypertensive diseases. Key factors have been implicated in leading to these complications including hyperglycemia, insulin resistance, dyslipidemia, advanced glycation end products, growth factors, inflammatory cytokines/chemokines and related increases in cellular oxidant stress (including mitochondrial) and endoplasmic reticulum stress. However, the molecular mechanisms underlying the high incidence of diabetic complications, which often progress despite glycemic control, are still not fully understood. MicroRNAs (miRNAs) are short non-coding RNAs that have elicited immense interest in recent years. They repress target gene expression via post-transcriptional mechanisms and have diverse cellular and biological functions. Herein, we have discussed the role of miRNAs in the pathobiology of various diabetic complications, their involvement in oxidant stress, and also the potential use of differentially expressed miRNAs as novel diagnostic biomarkers and therapeutic targets.

IntroductionBreast tumor kinase (Brk/protein tyrosine kinase 6 (PTK6)) is a nonreceptor, soluble tyrosine kinase overexpressed in the majority of breast tumors. Previous work has placed Brk downstream of epidermal growth factor receptor (ErbB) activation and upstream of extracellular signal-regulated kinase 5 (ERK5) and p38 mitogen-activated protein (MAP) kinases. Herein we investigate the regulation of Brk kinase activity and cell migration in response to treatment of keratinocytes (HaCaT cells) and breast cancer cell lines (MDA-MB-231 and T47D cells) with hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP), peptide ligands for Met and Ron receptors, respectively.MethodsIn vitro kinase assays were performed to directly measure Brk kinase activity in response to MET and RON ligands. Transfection of Brk-targeted RNAi was used to knock down endogenous Brk or ERK5 in multiple cell lines. Kinase activities (downstream of MET signaling) were assayed by Western blotting using total and phospho-specific antibodies. Boyden chamber assays were used to measure cell migration in response to manipulation of Brk and downstream MET effectors. Rescue experiments were performed by knock down of endogenous Brk using RNAi (targeting the untranslated region (3′-UTR)) and transient transfection (re-expression) of either wild-type or kinase-inactive Brk.ResultsBrk gene silencing revealed that HGF, but not MSP, induced robust Brk-dependent cell migration. Brk and ERK5 copurified in HGF-induced protein complexes, and Brk/ERK5 complexes formed independently of Brk kinase activity. ERK5 was required for breast cancer cell but not keratinocyte cell migration, which became ERK1/2-dependent upon ERK5 knockdown. Notably, rescue experiments indicated that the kinase activity of Brk was not required for HGF-induced cell migration. Further, expression of either wild-type or kinase-inactive Brk in Brk-null MDA-MB-435 cells activated ERK5 and conferred increased HGF-induced cell migration.ConclusionsThese results have identified Brk and ERK5 as important downstream effectors of Met signaling to cell migration. Targeting ERK5 kinase activity or inhibiting the formation of Brk/ERK5 complexes may provide an additional means of blocking cell migration associated with breast cancer progression to metastasis.

Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-β1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-β-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-β-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-β. TGF-β-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-β-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-β, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-β-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-β through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-β target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-β-induced collagen accumulation in DN.

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Increased TGF‐β1 expression in glomerular mesangial cells (MC) augments extracellular matrix accumulation, hypertrophy and pro‐fibrotic genes during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. microRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF‐β1 to enhance the expression of pro‐fibrotic genes like collagen. In this study, we found a significant decrease in the expression of key members of the miR‐130 and miR‐30 families in mouse MC (MMC) treated with TGF‐β1. In parallel there was a decrease in host genes 2610318N02RIK (RIK, miR‐130b host gene) and NF‐YC (miR‐30e* host gene), suggesting host gene‐dependent expression of these miRNAs. TGF‐β receptor1 (TGF‐βR1), was identified as a target of miR‐130b. Interestingly, the RIK promoter contains three NF‐YC binding sites and was regulated by NF‐YC, a potential target of miR‐216a which was also up‐regulated by TGF‐β1. Therefore, a novel cascade, ↑TGF‐β1>↑miR‐216a>↓NF‐YC>↓RIK (↓miR‐130b) may up‐regulate TGF‐βR1 to augment expression of TGF‐β1 target fibrotic genes. miR‐130 and miR‐30e* were down‐regulated, whereas TGF‐βR1, as well as the pro‐fibrotic genes COLIVa1, COLXIIa1, CTGF and PAI‐1 were up‐regulated by TGF‐β1 in MMC and in the glomeruli of streptozotocin injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA‐ and their host gene‐mediated cascade initiated by TGF‐β1 which results in the up‐regulation of pro‐fibrotic factors such as TGF‐βR1 and collagens associated with the progression of DN. Grant Funding Source: Supported by NIH R01 DK081705 and ADA 7‐10‐MI‐07

Signal transduction pathways downstream of receptor tyrosine kinases (RTKs) are often deregulated during oncogenesis, tumor progression, and metastasis. In particular, the peptide growth factor hormone, hepatocyte growth factor (HGF), and its specific receptor, Met tyrosine kinase, regulate cancer cell migration, thereby conferring an aggressive phenotype (Nakamura et al., J Clin Invest 106 (12):1511–1519, 2000; Huh et al., Proc Natl Acad Sci U S A 101:4477–4482, 2004). Additionally, overexpression of Met is associated with enhanced invasiveness of breast cancer cells (Edakuni et al., Pathol Int 51(3):172–178, 2001; Jin et al., Cancer 79(4):749–760, 1997; Tuck et al., Am J Pathol 148(1):225–232, 1996). Here, we review the regulation of recently identified novel downstream mediators of HGF/Met signaling, Breast tumor kinase (Brk/PTK6), and Src-associated substrate during mitosis of 68 kDa (Sam68), and discuss their relevance to mechanisms of breast cancer progression.

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Interactions between luminal epithelial cells and their surrounding microenvironment govern the normal development and function of the mammary gland. Alterations of these interactions can induce abnormal intracellular signaling pathways that affect the development and progression of breast tumors. One critical component of mammary gland development, as well as breast cancer progression, is the expression of estrogen receptors. In a previous study using cultured nonmalignant mammary epithelial cells, we found that the basement membrane molecules, laminin-1 and collagen-IV, were involved in maintenance of estrogen receptor (ER) alpha expression, and that this response could be interfered with by disrupting cell-extracellular matrix adhesion. Here we use phenotypically normal mammary epithelial SCp2 cells to dissect the promoter region of the ERalpha that is involved in the selective response to basement membrane. We also analyze the alteration of this response in SCg6 cells, a malignant cell line that shares a common lineage with the SCp2 cells, to provide insight into the relative overexpression of ERalpha and the unresponsiveness to basement membrane regulation found in those malignant cells. Evidence is presented to show the relevance of the cross-talk between different signaling pathways in the constitution of a functional tissue organization and how this integration may be disrupted in the malignant phenotype.