Induction of extracellular matrix gene expression in normal human keratinocytes by transforming growth factor beta is altered by cellular differentiation

Changes in epithelial substrate have been related to the cellular capacity for proliferation and to changes in cellular behavior. The effect of TGF beta 1 on the expression of the basement membrane genes, fibronectin, laminin B1, and collagen alpha 1 (IV), was examined. Northern analysis revealed that treatment of normal human epidermal keratinocytes with 100 pM TGF beta 1 increased the expression of each extracellular matrix (ECM) gene within 4 h of treatment. Maximal induction was reached within 24 h after treatment. The induction of ECM mRNA expression was dose dependent and was observed at doses as low as 1-3 pM TGF beta 1. Incremental doses of TGF beta 1 also increased cellular levels of fibronectin protein in undifferentiated keratinocytes and resulted in increased secretion of fibronectin. Squamous-differentiated cultures of keratinocytes expressed lower levels of the extracellular matrix RNAs than did undifferentiated cells. Treatment of these differentiated cells with TGF beta 1 induced the expression of fibronectin mRNA to levels seen in TGF beta-treated, undifferentiated keratinocytes but only marginally increased the expression of collagen alpha 1 (IV) and laminin B1 mRNA. The increased fibronectin mRNA expression in the differentiated keratinocytes was also reflected by increased accumulation of cellular and secreted fibronectin protein. The inclusion of cycloheximide in the protocol indicated that TGF beta induction of collagen alpha 1 (IV) mRNA was signaled by proteins already present in the cells but that TGF beta required the synthesis of a protein(s) to fully induce expression of fibronectin and laminin B1 mRNA. The differential regulation of these genes in differentiated cells may be important to TGF beta action in regulating reepithelialization.     

Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue

Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by quality control experiments to obtain cells that can be used for further investigations. After different steps of digestion of the tissue using collagenase, DNase and dispase, the resulting cells are purified using Percoll gradient. The success of the isolation can be analyzed by cell viability test (Trypan Blue exclusion test) and by flow cytometric analysis to assess apoptosis. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA expression, cell-proliferation assay or protein analysis. This protocol can be completed within 6-7 h.     

Detection of DNA fragmentation and apoptotic proteins, and quantification of uncoupling protein expression by real-time RT-PCR in adipose tissue

Better understanding of the mechanisms involved in adipose tissue growth and metabolism is critical for the development of more effective treatments for obesity. However, because of its high lipid and low protein content, adipose tissue can present unique problems in some experimental procedures. We describe three protocols that provide new or improved methods for analysis of DNA, RNA, and protein from different adipose tissues. The first protocol provides a simple and rapid method for separation of fragmented DNA and visualization of apoptotic DNA laddering without the need for radioisotopes. This technique allows for an estimate of the amount of DNA fragmentation, and hence, apoptosis. The second protocol details subcellular fractionation of adipose tissue for the extraction of protein in the mitochondrial and cytosol fractions and the measurement of apoptotic protein (Bcl-2 and Bax) levels in each fraction. The last protocol involves extraction of total RNA from adipose tissue and the measurement of uncoupling protein mRNA using real-time RT-PCR, a method that has not previously been used to measure expression of uncoupling proteins in adipose tissue.     

Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues

Fresh or frozen tissue samples will always be the best tissue source for the analysis of nucleic acids and proteins from tissues. However, their long-term storage is expensive and laborious. Much interest has therefore been focused on the question whether the almost infinite resources of formalin fixed and paraffin embedded tissue samples in the archives of pathology and histology departments can be used for research on biomarkers and molecular mechanisms of disease. In recent years the methods and protocols for the extraction of DNA, mRNA, miRNA and proteins from formalin-fixed and paraffin-embedded tissue samples have improved enormously. Especially, the possibilities of analysing DNA and miRNA in FFPE have reached a level that allows their application as a first line approach in the search for biomarkers. In contrast, many questions remain in terms of quantification of mRNA and protein expression levels in formalin-fixed and paraffin-embedded tissue samples. This review gives an overview on current potentials and limitations of the quantification of DNA, miRNA, mRNA and the proteome in FFPE tissue samples. The chemical events during formalin fixation and paraffin embedding and alternatives to formalin fixation are described. In addition, methods and general problems of DNA, miRNA, mRNA and protein extraction and the current knowledge on the feasibility and accuracy of quantitative gene expression analysis in FFPE tissues is summarized.     

Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells

A 21-base pair RNA duplex that perfectly matches an endogenous target mRNA selectively degrades the mRNA and suppresses gene expression in mammalian tissue culture cells. A single base mismatch with the target is believed to protect the mRNA from degradation, making this type of interference highly specific to the targeted gene. A short RNA with mismatches to a target sequence present in multiple copies in the 3'-untranslated region of an exogenously expressed gene can, however, silence it by translational repression. Here we report that a mismatched RNA, targeted to a single site in the coding sequence of an endogenous gene, can efficiently silence gene expression by repressing translation. The antisense strand of such a mismatched RNA requires a 5'-phosphate but not a 3'-hydroxyl group. G.U wobble base pairing is tolerated as a match for both RNA degradation and translation repression. Together, these findings suggest that a small inhibitory RNA duplex can suppress expression of off-target cellular proteins by RNA degradation or translation repression. Proper design of experimental small inhibitory RNAs or a search for targets of endogenous micro-RNAs must therefore take into account that these short RNAs can affect expression of cellular genes with as many as 3-4 base mismatches and additional G.U mismatches.     

Enhanced matrix synthesis in de novo, scaffold free cartilage-like tissue subjected to compression and shear

Production of a de novo cartilage-like tissue construct is a goal for the repair of traumatic chondral defects. We aimed to enhance the matrix synthesis within a scaffold free, de novo cartilage-like tissue construct by way of mechanical load. A novel loading machine that enables the application of shear, as well as compression, was used to subject tissue engineered cartilage-like tissue to mechanical stress. The machine, which applies the load through a roller mechanism, can load up to 20 constructs with four different loading patterns simultaneously. The expression of mRNA encoding matrix products, and subsequent changes in matrix protein content, were analyzed after various loading regimes. The force applied to the immature tissue had a direct bearing on the short-term (first 4 h) response. A load of 0.5 N caused an increase in collagen II and aggrecan mRNA within an hour, with a peak at 2 h. This increased mRNA expression was translated into an increase of up to 60% in the glycosaminoglycan content of the optimally loaded constructs after 4 days of intermittent cyclical loading. Introducing pauses between load cycles reproducibly lead to an increase in GAG/DNA. In contrast, constant cyclical load, with no pause, lead to a decrease in the final glycosaminoglycan content compared with unloaded controls. Our data suggest that a protocol of mechanical stimulation, simulating in vivo conditions and involving shear and compression, may be a useful mechanism to enhance the properties of tissue engineered tissue prior to implantation.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.     

Effect of hypertension on fibronectin expression in the rat aorta

Interactions between extracellular fibronectin and vascular cells are thought to influence the phenotype of those cells. To determine if changes in fibronectin expression accompany the phenotypic changes of vascular tissue characteristic of experimental hypertension, steady state mRNA levels for fibronectin were determined in aortae of normotensive and hypertensive rats. A 3-6-fold increase in fibronectin mRNA was observed in aortic tissue of hypertensive rats following 3 weeks of treatment with deoxycorticosterone and salt, whereas if rats were treated only with deoxycorticosterone or salt alone, no changes occurred. The changes were reversed by normalization of blood pressure. The increases observed were localized to aorta and not to the periaortic tissue. Angiotensin II infusion using osmotic minipumps also caused an increase in fibronectin expression. Age-dependent increases in aortic fibronectin mRNA occurred in several rat strains, and the combined effects of hypertension and aging were greater than either variable alone. A clear distinction between the expression of fibronectin mRNA and that for collagen or tropoelastin were found in hypertensive and aging models. Aortic fibronectin was also increased in the hypertensive rats as determined by Western blot analysis. The findings indicate that elevation in blood pressure increases fibronectin expression in rat aorta and suggest that such changes may influence the aortic cellular responses to hypertension.     

Histological aspects of in situ hybridization. Detection of poly(A) nucleotide sequences in mouse liver sections as a model system

This study examined the detection of cellular poly(A) sequences in mouse liver sections by in situ hybridization using a 3H-labelled poly(dT) probe. Parameters examined included possible losses of target poly(A) sequences from sectioned cells, access of probe to target sequences, section thickness, hybridization conditions, autoradiographic efficiency, specific activity of probes and specificity of reaction. An improved protocol was devised that resulted in good preservation of histological detail in sectioned tissue blocks, and a calculated hybridization efficiency of 50%-100%. With the use of probes of defined sequence, the protocol should allow detection of unique mRNA sequences within single cells with an estimated sensitivity of 6-12 unique mRNA molecules per sectioned cell.     

Tissue-specific expression of insulin receptor isoforms in obesity/type 2 diabetes mouse models

The two insulin receptor (IR) isoforms IR-A and IR-B are responsible for the pleiotropic actions of insulin and insulin-like growth factors. Consequently, changes in IR isoform expression and in the bioavailability of their ligands will impact on IR-mediated functions. Although alteration of IR isoform expression has been linked to insulin resistance, knowledge of IR isoform expression and mechanisms underlying tissue/cell-type-specific changes in metabolic disease are lacking. Using mouse models of obesity/diabetes and measuring the mRNA of the IR isoforms and mRNA/protein levels of total IR, we provide a data set of IR isoform expression pattern that documents changes in a tissue-dependent manner. Combining tissue fractionation and a new in situ mRNA hybridization technology to visualize the IR isoforms at cellular resolution, we explored the mechanism underlying the change in IR isoform expression in perigonadal adipose tissue, which is mainly caused by tissue remodelling, rather than by a shift in IR alternative splicing in a particular cell type, e.g. adipocytes.     

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.     

Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways.

Vascular endothelial growth factor (VEGF) is an angiogenic polypeptide that has been isolated from a variety of tumorigenic and nontransformed cell lines. Because of the importance of blood vessel growth to cell and tissue development, we have examined VEGF gene expression in a variety of mouse tissues and rodent models of cellular differentiation. Using a cloned murine VEGF cDNA we show that VEGF mRNA is expressed at relatively low levels in many adult mouse tissues examined. However, this message is dramatically induced in two models of cell differentiation: 3T3-adipose conversion and C2C12 myogenic differentiation. VEGF protein secretion is also induced in adipocyte differentiation. VEGF mRNA is markedly regulated in a pheochromocytoma (PC12) cell model of transformation and differentiation. The transformed undifferentiated cells express moderate levels of VEGF mRNA and this expression is virtually extinguished when cells differentiate into non-malignant neuron-like cells. Experiments employing phorbol esters and cAMP analogues indicate that VEGF mRNA expression is stimulated in preadipocytes by both protein kinase C and protein kinase A-mediated pathways. These results suggest that VEGF mRNA levels are closely linked to the process of cellular differentiation; they also clearly demonstrate that expression of this angiogenic factor is specifically regulated in a transformed cell line, possibly via aberrant activation of cellular second messenger pathways.     

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.