MicroRNA-143 regulates adipocyte differentiation

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.     

MicroRNA-145-based differentiation of human mesenchymal stem cells to smooth muscle cells

Objectives

To investigate the role of microRNA-145, that regulates gene expression of genes related to differentiation, proliferation and the phenotype of smooth muscle cells (SMCs), in the differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) to SMCs.

Results

Real-time PCR analysis indicated significant upregulation of SMC markers, including SM-α-actin, calponin, caldesmon and SMMHC, in SMCs compared to hBM-MSCs. Conversely, Krüppel-like factor 4, the direct target of microRNA-145 and the suppressor of smooth muscle differentiation, was suppressed in hBM-MSC-derived SMCs. Western blot analysis and immunocytochemistry also confirmed that the introduction of microRNA-145 into hBM-MSCs induced mature contractile SMCs. The functionality of hBM-MSC-derived SMCs was assessed by proliferation assay using PDGF-BB and contractility assay using carbachol. The results showed that the produced SMCs contracted in response to carbachol stimulation.

Conclusion

Overexpression of microRNA-145 in undifferentiated hBM-MSCs results in functionally mature contractile SMCs that can be used in drug discovery and cell therapy in SMC disorders such as vascular disease.

     

MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2

Elucidation of the molecular mechanisms governing human adipose-derived mesenchymal stem cells (hASCs) osteogenic differentiation is of great importance for improving the treatment of bone-related diseases. In this study, we examined the role of microRNA (miR)-100 on the osteogenesis of hASCs. Overexpression of miR-100 inhibited osteogenic differentiation of hASCs in vitro, whereas downregulation of miR-100 enhanced the process. Target prediction analysis and dual luciferase report assay confirmed that bone morphogenetic protein receptor type II (BMPR2) was a direct target of miR-100. Furthermore, knockdown of BMPR2 by RNA interference inhibited osteogenic differentiation of hASCs, similar as the effect of upregulation miR-100. Taken together, our findings imply that miR-100 plays a negative role in osteogenic differentiation and might act through targeting BMPR2.     

Sonic hedgehog regulates prostatic growth and epithelial differentiation

The Sonic hedgehog (SHH)-signalling pathway mediates epithelial-mesenchymal interactions in several tissues during development and disease, and we have investigated its role in rat ventral prostate (VP) development. We have demonstrated that Shh and Ptc expression correlates with growth and development of the prostate and that their expression is not regulated by androgens in the VP. Prostatic budding was induced in response to testosterone in Shh null mouse urogenital sinus (UGS) explants grown in vitro and in rat UGS explants cultured with cyclopamine, suggesting that SHH-signalling is not critical for prostatic induction. SHH-signalling was disrupted at later stages of VP development (in vitro), resulting in a reduction in organ size, an increase in ductal tip number, and reduced proliferation of ductal tip epithelia. The addition of recombinant SHH to VPs grown in vitro caused a decrease in ductal tip number and expansion of the mesenchyme. In the presence of testosterone, inhibition of SHH-signalling accelerated the canalisation of prostatic epithelial ducts and resulted in ducts that showed morphological similarities to cribiform prostatic intraepithelial neoplasia (PIN). The epithelia of these ducts also demonstrated precocious and aberrant differentiation, when examined by immunohistochemistry for p63 and cytokeratin 14. In conclusion, we show that SHH-signalling is not essential for prostatic induction, but is important for prostatic growth, branching, and proliferation, and that androgen-stimulated growth in the absence of signalling from the SHH pathway results in aberrant epithelial differentiation.     

Basement membrane heterogeneity and variation in corneal epithelial differentiation

We have previously shown that the expression of a major 64-Kda keratin (K3) in corneal epithelium is site-related. It is found suprabasally in limbal epithelium, but uniformly (basal cells included) in central corneal epithelium. In the present study, we used a panel of antibodies against various components of corneal epithelial basement membrane to investigate a possible correlation between basement membrane heterogeneity and differential (basal vs. suprabasal) K3 keratin expression. One of these antibodies, AE27, stains human conjunctival basement membrane weakly, limbal basement membrane heterogeneously, and central corneal basement membrane strongly. Basal cells resting on basement membrane that stains strongly with AE27 tend to stain with monoclonal antibody AE5, which recognizes keratin K3. Basal cells on basement membrane staining weakly with AE27 tend not to stain with AE5. No such correlation exists between AE5 staining and type IV collagen, which is detectable immunohistochemically in conjunctival and limbal basement membrane, but not in corneal basement membrane overlying Bowman's layer. These results suggest that basement membrane of human corneal/conjunctival epithelium can be divided into at least three domains: the conjunctival basement membrane (type IV collagen-positive, AE27-weak), the limbal basement membrane (type IV collagen-positive, AE27-strong), and corneal basement membrane (type IV collagen-negative, AE27-strong). The results also raise the possibility that basement membrane heterogeneity may play a functional role in regulating keratin expression and other aspects of differentiation of corneal epithelium; more experiments are needed to test this hypothesis.     

Beta-catenin regulates differentiation of respiratory epithelial cells in vivo

An activated form of beta-catenin [Catnb(Delta(ex3))] was expressed in respiratory epithelial cells of the developing lung. Although morphogenesis was not altered at birth, air space enlargement and epithelial cell dysplasia were observed in the early postnatal period and persisted into adulthood. The Catnb(Delta(ex3)) protein caused squamous, cuboidal, and goblet cell dysplasia in intrapulmonary conducting airways. Atypical epithelial cells that stained for surfactant pro protein C (pro-SP-C) and had morphological characteristics of alveolar type II cells were observed in bronchioles of the transgenic mice. Catnb(Delta(ex3)) inhibited expression of Foxa2 and caused goblet cell hyperplasia associated with increased staining for mucins and the MUC5A/C protein. In vitro, both wild type and activated beta-catenin negatively regulated the expression of the Foxa2 promoter. Catnb(Delta(ex3)) also caused pulmonary tumors in adult mice. Activation of beta-catenin caused ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, demonstrating a critical role for the Wnt/beta-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung.     

Oxygen tension affects terminal differentiation of corneal limbal epithelial cells

Oxygen concentration has been shown to be crucial in the proliferation and differentiation of various types of cells, while the impact of oxygen tension on the lineage commitment of epithelial cells remains elusive. In this study, we investigated the effect of hypoxia on the differentiation of corneal limbal epithelium using an ex vivo squamous metaplasia model. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of human corneal limbal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The Notch signaling pathway was activated in hypoxic cultures, whereas the p38 MAPK signaling pathway was downregulated. The addition of Notch inhibitor under hypoxic conditions restored the activation of p38 MAPK and resulted in the recidivation of limbal epithelial cells to epidermal-like differentiation. Moreover, the epidermal-like differentiation of rabbit limbal epithelial cells was also blocked under hypoxic conditions in corneal epithelial cell sheets engineered ex vivo. We concluded that hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal limbal epithelial cells. Hypoxia coupled with air exposure can be used in the tissue engineering of corneal limbal epithelium.     

CDK5 regulates cell adhesion and migration in corneal epithelial cells

CDK5 and its activator, p35, are expressed in mouse corneal epithelium and can be coimmunoprecipited from corneal epithelial cell lysates. Immunostaining shows CDK5 and p35 in all layers of the corneal epithelium, especially along the basal side of the basal cells. Stable transfection of corneal epithelial cells with CDK5, which increases CDK5 kinase activity by approximately 33%, also increases the number of cells adhering to fibronectin and the strength of adhesion. CDK5 kinase activity seems to be required for this effect, because the kinase inactive mutation, CDK5-T33, either reduces adhesion or has no significant effect, depending on the level of expression. Using an in vitro scrape wound in confluent cultures of stably transfected cells to examine the effect of CDK5 on cell migration, we show that reoccupation of the wound area is significantly decreased by CDK5 and increased by CDK5-T33. These findings indicate that CDK5 may be an important regulator of adhesion and migration of corneal epithelial cells.     

MicroRNA-143 and -145 in colon cancer

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs (20-22 nucleotides) that negatively regulate gene expression at the translational level by base pairing to the 3' untranslated region of target messenger RNAs. More than 400 miRNAs have been identified in humans and are evolutionally conserved from plants to animals. It has been revealed that miRNAs regulate various biological processes, such as development, cell differentiation, cell proliferation, and cell death. It is predicted that 30% of protein-encoding genes are regulated by miRNAs. Inappropriate expression of miRNAs has been found in cancer. Especially, the expression level of miRNAs that act like anti-oncogenes is frequently reduced in cancers because of chromosome aberrations. In addition, since the processing of miRNAs has been characterized to be enzymatic in nature, the expression levels of miRNAs are closely associated with the activity and levels of such enzymes. In this review, we discuss recent remarkable advances in miRNA biogenesis, bio-networking involving miRNAs, and their roles in carcinogenesis. Further, we discuss the expression of miRNA-143 and -145 in colon cancer and their roles in carcinogenesis. The available data suggest that miRNAs would be potentially useful as diagnostic and therapeutic tools.     

The smooth muscle microRNA miR-145 regulates gut epithelial development via a paracrine mechanism

MicroRNAs are potent modulators of cellular differentiation. miR-145 is expressed in, and promotes the differentiation of vascular and visceral smooth muscle cells (SMCs). Interestingly, we have observed that miR-145 also promotes differentiation of the gut epithelium in the developing zebrafish, a cell type where it is not expressed. Here we identify that a paracrine pathway involving the morphogens Sonic hedgehog (Shh) in epithelium and bone morphogenic protein 4 (Bmp4) in SMCs is modulated by miR-145. We show that expression of miR-145 in visceral SMCs normally represses the expression of the morphogen bmp4, as loss of miR-145 leads to upregulation of bmp4 in SMCs. We show that bmp4 in turn controls expression of Shh in the visceral epithelium. Conversely, in miR-145 morphants where bmp4 expression is increased, expression of sonic hedgehog a (shha) is strongly increased in gut epithelium. We show that expression of bmp4 is modulated by the miR-145 direct target gata6 but not a second potential direct target, klf5a. Thus although miR-145 is a tissue-restricted microRNA, it plays an essential role in promoting the patterning of both gut layers during gut development via a paracrine mechanism.     

MicroRNA-145 directly targets the insulin-like growth factor receptor I in human bladder cancer cells

The insulin-like growth factor receptor I (IGF-IR) is a proto-oncogene with potent mitogenic and antiapoptotic activities. It has been reported that expression of IGF-IR is up-regulated in bladder cancer. Here, we assessed whether microRNA-145 (miR-145) regulates IGF-IR expression in bladder cancer. In our study, miR-145 was shown to directly target IGF-IR 3′-untranslated region (UTR) in human bladder cancer cells. Small interfering RNA (siRNA)- and miR-145-mediated IGF-IR knockdown experiments revealed that miR-145 promotes cell apoptosis, and suppresses cell proliferation and migration through suppression of IGF-IR expression. Taken together, our data suggest that miR-145 may inhibit bladder cancer initiation by affecting IGF-IR signaling.     

Mesenchymal reprogramming of adult human epithelial differentiation

The objective of this study was to determine whether neonatal rat seminal vesicle mesenchyme (rSVM) can reprogram epithelial differentiation in a fully differentiated adult human bladder epithelium. For this purpose neonatal rSVM was isolated from newborn (0-day) Sprague-Dawley rats, and normal adult human bladder epithelium (hBLE) was isolated from radical cystoprostatectomy specimens to prepare rSVM+hBLE tissue recombinants in vitro. After overnight culture the tissue recombinants were grafted beneath the renal capsule of male athymic rodent hosts and allowed to grow in vivo for 6 months. As controls, rSVM and hBLE were grafted separately and allowed to grow for the same period. Tissue recombinants and control tissue grafts were harvested, and secretions were collected for biochemical studies. Tissues were fixed both for histologic as well as immunohistochemical staining. Neonatal rSVM induced normal adult human bladder urothelium to form glandular structures resembling prostate. The induced prostatic acini were filled with secretions that expressed human prostate-specific secretory proteins. These findings demonstrate that adult human urothelial cells retain a responsiveness to neonatal prostatic mesenchymal inductors. Change in urothelial histodifferentiation was associated with change in functional activity. The ability of the neonatal rat mesenchymal tissues to induce morphologic as well as biochemical changes in normal adult human urothelium provides a basis for human tissue engineering and organ reconstruction.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.