Tissue inhibitor of metalloproteinase-1 (TIMP-1) regulates mesenchymal stem cells through let-7f microRNA and Wnt/ß-catenin signaling.

Tissue inhibitor of metalloproteinases 1 (TIMP-1) is a matrix metalloproteinase (MMP)-independent regulator of growth and apoptosis in various cell types. The receptors and signaling pathways that are involved in the growth factor activities of TIMP-1, however, remain controversial. RNA interference of TIMP-1 has revealed that endogenous TIMP-1 suppresses the proliferation, metabolic activity, and osteogenic differentiation capacity of human mesenchymal stem cells (hMSCs). The knockdown of TIMP-1 in hMSCs activated the Wnt/ß-catenin signaling pathway as indicated by the increased stability and nuclear localization of ß-catenin in TIMP-1-deficient hMSCs. Moreover, TIMP-1 knockdown cells exhibited enhanced ß-catenin transcriptional activity, determined by Wnt/ß-catenin target gene expression analysis and a luciferase-based ß-catenin-activated reporter assay. An analysis of a mutant form of TIMP-1 that cannot inhibit MMP indicated that the effect of TIMP-1 on ß-catenin signaling is MMP independent. Furthermore, the binding of CD63 to TIMP-1 on the surface of hMSCs is essential for the TIMP-1-mediated effects on Wnt/ß-catenin signaling. An array analysis of microRNAs (miRNAs) and transfection studies with specific miRNA inhibitors and mimics showed that let-7f miRNA is crucial for the regulation of ß-catenin activity and osteogenic differentiation by TIMP-1. Let-7f was up-regulated in TIMP-1-depleted hMSCs and demonstrably reduced axin 2, an antagonist of ß-catenin stability. Our results demonstrate that TIMP-1 is a direct regulator of hMSC functions and reveal a regulatory network in which let-7f modulates Wnt/ß-catenin activity.

MicroRNA-126, -145, and -155: a therapeutic triad in atherosclerosis?

Atherosclerosis is a condition caused by lipid-induced inflammation of the vessel wall orchestrated by a complex interplay of various cell types, such as endothelial cells, smooth muscle cells, and macrophages. MicroRNAs (miRNAs) have emerged as key regulators of gene expression typically by repressing the target mRNA, which determines cell fate and function under homeostatic and disease conditions. Here, we outline the effects of miRNA-145, -126, and -155 in atherosclerosis in vivo. Downregulation of miR-145, which controls differentiation of smooth muscle cells, promotes lesion formation, whereas the endothelial cell-specific miRNA-126 signals the need for endothelial repair through its transfer from apoptotic endothelial cells in microvesicles. Elevated miR-155 levels are characteristic of proinflammatory macrophages and atherosclerotic lesions. However, the effects of miR-155 seem to be different in early and advanced atherosclerosis. The discovery of the role of these miRNAs in atherosclerosis sheds light on the current concepts of atherogenesis and may provide novel treatment options for cardiovascular diseases.

Blocking the proliferation of human tumor cell lines by peptidase inhibitors from Bauhinia seeds.

In cancer tumors, growth, invasion, and formation of metastasis at a secondary site play a pivotal role, participating in diverse processes in the development of the pathology, such as degradation of extracellular matrix. Bauhinia seeds contain relatively large quantities of peptidase inhibitors, and two Bauhinia inhibitors were obtained in a recombinant form from the Bauhinia bauhinioides species, B. bauhinoides cruzipain inhibitor, which is a cysteine and serine peptidase inhibitor, and B. bauhinioides kallikrein inhibitor, which is a serine peptidase inhibitor. While recombinant B. bauhinoides cruzipain inhibitor inhibits human neutrophil elastase cathepsin G and the cysteine proteinase cathepsin L, recombinant B. bauhinioides kallikrein inhibitor inhibits plasma kallikrein and plasmin. The effects of recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor on the viability of tumor cell lines with a distinct potential of growth from the same tissue were compared to those of the clinical cytotoxic drug 5-fluorouracil. At 12.5 µM concentration, recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor were more efficient than 5-fluorouracil in inhibiting MKN-28 and Hs746T (gastric), HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), and THP-1 and K562 (leukemia) cell lines. Additionally, recombinant B. bauhinoides cruzipain inhibitor inhibited 40 % of the migration of Hs746T, the most invasive gastric cell line, while recombinant B. bauhinioides kallikrein inhibitor did not affect cell migration. Recombinant B. bauhinioides kallikrein inhibitor and recombinant B. bauhinoides cruzipain inhibitor, even at high doses, did not affect hMSC proliferation while 5-fluorouracil greatly reduced the proliferation rates of hMSCs. Therefore, both recombinant B. bauhinoides cruzipain inhibitor and recombinant B. bauhinioides kallikrein inhibitor might be considered for further studies to block peptidase activities in order to target specific peptidase-mediated growth and invasion characteristics of individual tumors, mainly in patients resistant to 5-fluorouracil chemotherapy.

Dissecting the impact of Frizzled receptors in Wnt/ß-catenin signaling of human mesenchymal stem cells.

Wnt/ß-catenin signaling is of fundamental importance in the regulation of self-renewal, migration/invasion, and differentiation of human mesenchymal stem cells (hMSCs). Because little information is available about the function of Frizzled receptors (Fzds) as the main receptors of Wnt proteins in hMSCs, we first performed comparative Fzd mRNA expression profiling. Fzd9 and Fzd10 were not expressed in hMSCs. While Fzd3 was expressed at low levels in hMSCs, the other Fzds exhibited high expression rates. Activation and repression of Wnt signaling in hMSCs revealed that the expression levels of Fzd1, Fzd6, and Fzd7 are positively correlated with the Wnt/ß-catenin activation status, whereas Fzd8 exhibited an inverse relation. For studying the functional relevance of Fzds in Wnt/ß-catenin signaling, RNA interference, ectopic expression studies, and rescue approaches were performed in hMSCs carrying a highly sensitive TCF/LEF reporter gene system (Gaussia luciferase). We found that, Fzd1, Fzd5, Fzd7, and Fzd8 are largely involved in Wnt/ß-catenin signaling of hMSCs. Moreover, the knockdown of Fzd5 can be compensated by the ectopic expression of Fzd7. Conversely, the ectopic expression of Fzd5 in Fzd7-knockdown hMSCs resulted in a rescue of Wnt/ß-catenin signaling, pointing to a functional redundancy of Fzd5 and Fzd7.

Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways.

WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the ß-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced ß-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the ß-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.

The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines.

Supplementary to the efficient inhibition of trypsin, chymotrypsin, plasma kallikrein, and plasmin already described by the EcTI inhibitor from Enterolobium contortisiliquum, it also blocks human neutrophil elastase (K(iapp)=4.3 nM) and prevents phorbol ester (PMA)-stimulated activation of matrix metalloproteinase (MMP)-2 probably via interference with membrane-type 1 (MT1)-MMP. Moreover, plasminogen-induced activation of proMMP-9 and processing of active MMP-2 was also inhibited. Furthermore, the effect of EcTI on the human cancer cell lines HCT116 and HT29 (colorectal), SkBr-3 and MCF-7 (breast), K562 and THP-1 (leukemia), as well as on human primary fibroblasts and human mesenchymal stem cells (hMSCs) was studied. EcTI inhibited in a concentration range of 1.0-2.5 µM rather specifically tumor cell viability without targeting primary fibroblasts and hMSCs. Taken together, our data indicate that the polyspecific proteinase inhibitor EcTI prevents proMMP activation and is cytotoxic against tumor cells without affecting normal tissue remodeling fibroblasts or regenerative hMSCs being an important tool in the studies of tumor cell development and dissemination.

Wnt signalling in mouse mesenchymal stem cells: impact on proliferation, invasion and MMP expression.

Based on the capacity of mesenchymal stem cells (MSC) to differentiate into multiple cell types in vitro and in vivo, MCS may be a suitable source for cell therapy and regeneration strategies. A prerequisite for effective clinical applications of human MSC (hMSC) is a profound knowledge of signal transduction cascades that mediate processes like proliferation, targeted migration and differentiation. Recently, we identified the canonical Wnt signal transduction pathway as a key player in hMSC proliferation and invasion. To evaluate whether those findings are transferable to the equivalent counterparts in mice, we studied important steps in the wingless/int-1 (Wnt) signal transduction pathway in mouse MSC (mMSC) and mMSC carrying a T cell specific transcription factor (TCF)/lymphoid enhancer binding factor (LEF)-reporter transgene. We found that the induction of the canonical Wnt pathway resulted in the up-regulation of the known Wnt target gene cyclin D1, closely associated with an enhanced proliferation capacity of mMSC. Interestingly, the expression of the Wnt target gene membrane type 1-matrix metalloproteinase (MT1-MMP) was diminished in mMSC upon Wnt3a stimulation, which came along with an impaired invasion. In line with these findings, MMP-2 and MMP-9 expression levels in mMSC were also decreased after Wnt3a treatment. In contrast, inhibition of Wnt signalling by the knockdown of the transcriptional activator beta-catenin resulted in an up-regulation of MT1-MMP and mMSC invasion. By comparing these findings with the settings in hMSC, major differences in Wnt-regulated MMP expression were observed in mMSC. Thus, our data advice caution when mouse model systems represent the pre-clinical validation of MSC-mediated therapeutical approaches.

The Wnt signal transduction pathway in stem cells and cancer cells: influence on cellular invasion.

The regulative network conducting adult stem cells in endogenous tissue repair is of prime interest for understanding organ regeneration as well as preventing degenerative and malignant diseases. One major signal transduction pathway which is involved in the control of these (patho)physiological processes is the Wnt pathway. Recent results obtained in our laboratories showed for the first time that canonical Wnt signaling is critically involved in the control of the migration/invasion behaviour of human mesenchymal stem cells (hMSC). In the first part of this review, we describe that the regenerative state is closely linked to the activation of the Wnt pathway. Central hallmarks of activated stem cells are recapitulated in a similar way also in cancer metastasis, where the acquisition of an invasive cancer stem cell phenotype is associated with the induction of Wnt-mediated epithelial to mesenchymal transition (EMT). In the second part, the influence of proinflammatory cytokines such as transforming growth factor (TGF-)beta1, interleukin (Il-)1beta, and tumor necrosis factor (TNF-)alpha is discussed with regard to the invasive characteristics of hMSC. In this context, special attention has been paid on the role of matrix metalloproteinases (MMPs), such as MMP-2, MMP-9 and membrane type 1 (MT1)-MMP, as well as on the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2. Putative cross-talks between different signal transduction pathways that may amplify the invasive capacity of this stem cell population are also discussed. Finally, the consequences towards future drug-mediated therapeutical modifications of Wnt signaling in stem cells and tumor cells are highlighted.

Genome-wide monocytic mRNA expression in polytrauma patients for identification of clinical outcome.

Immune activation in multiple trauma is closely linked to the development of multiple organ dysfunction and failure, and consequently, has a profound influence on patient outcome. Although peripheral blood monocytes play a critical role in this immune response, the biological significance of changes in genome-wide expression immediately after traumatic injury have not been explored previously. Thirteen patients presenting with multiple blunt trauma were studied. Peripheral blood monocytes were obtained within 90 min and at 6, 12, 24, 48, and 72 h after trauma. Apparent genome-wide expression was determined with Affymetrix U133A microarrays. Supervised analysis identified 698 probe sets that were differentially expressed in the 13 trauma subjects (P < 0.001) over the 72-h study period. An additional 763 probe sets were differentially expressed in patients who died (n = 3) compared with those who survived (n = 10). The ability of these probe sets to function as a classifier of survival was significantly demonstrated with six prediction models. Using pathway analysis, a network of proinflammatory genes and intracellular signaling pathways leading to c-JUN activation were consistently overexpressed in patients who died. Genome-wide mRNA expression patterns in circulating peripheral blood monocytes from multiple-injured patients can discriminate clinical outcome. The pattern of gene expression in patients who died suggests that in these individuals, there is a reprioritization of gene expression consistent with an early activation of selected genes involved in the initiation and propagation of a proinflammatory response.

Initial posttraumatic translocation of NF-kappaB and TNF-alpha mRNA expression in peripheral blood monocytes of trauma patients with multiple injuries: a pilot study.

Post-traumatic inflammation is connected to monocyte dysfunction characterized by reduced NF-kappaB translocation during the first post-traumatic days. Because the exact dynamic of monocytic NF-kappaB translocation in patients directly after trauma remains unclear, the aim of this pilot study was to measure the intranuclear presence of NF-kappaB in monocytes from patients with multiple injuries initially after the trauma and during the early post-traumatic period and to compare these results with downstream-placed mRNA expression alteration of TNF-alpha, as well as with clinical data. Eleven patients were enrolled with an Injury Severity Score of 16 to 66 points, and blood samples were drawn on admission within 90 min and at 6, 12, 24, 48, and 72 h after trauma. NF-kappaB translocation of monocytic nuclear protein was analyzed by electrophoretic mobility shift assay and was quantified by densitometry as arbitrary units. In addition, monocytes of healthy volunteers were analyzed either native (-, control) or after LPS stimulation (+, control). For determination of downstream mRNA encoding for TNF-alpha, quantitative reverse transcriptase-PCR was performed. For both parameters, the negative control values were set as baseline (=1) and results from positive controls and patients were given as a relative alteration ratio without unit. Initial post-traumatic NF-kappaB translocation was significantly increased in trauma patients on admission (88 +/- 37) and 6 h after trauma (59 +/- 28) compared with the baseline level. In contrast, TNF-alpha mRNA was not increased on admission (1.7 +/- 0.9) and decreased even below baseline after 12 h. The substantial information of our study arises from the analysis of the dynamic of NF-kappaB translocation of monocytes. Enabled by closely matched sequential blood sampling strictly standardized to the traumatic event, an essential increase of monocytic signal transduction and transcription could be elucidated in the very early post-traumatic period, which precedes the down-regulation of the innate immune system.

Characterization of osteosarcoma cell lines MG-63, Saos-2 and U-2 OS in comparison to human osteoblasts.

BACKGROUND: Osteosarcomas are malignant bone tumors consisting of cells with abnormal cellular functions. Although osteosarcoma-derived cells are commonly used for osteoblastic models, the molecular composition of the osteosarcoma extracellular matrix (ECM) is not well characterized. MATERIALS AND METHODS: We compared three osteosarcoma cell lines (MG-63, Saos-2 and U-2 OS) with normal human osteoblasts by immunocytochemistry. Cellular characteristics were assessed by morphometric analysis and proliferation kinetics. RESULTS: All investigated osteosarcoma cell lines exhibited very heterogeneous labelling profiles and each differed significantly from that of normal osteoblasts. Saos-2 cells revealed the most mature osteoblastic labelling profile while U-2 OS cells were negative for most of the investigated osteoblastic markers. CONCLUSION: We conclude that each osteosarcoma cell line exhibits a characteristic labelling profile and thus produces a differently composed extracellular matrix. This can be used in attempts to better characterize osteosarcoma, a as well as for their diagnosis.

ß-Catenin-dependent pathway activation by both promiscuous "canonical" WNT3a-, and specific "noncanonical" WNT4- and WNT5a-FZD receptor combinations with strong differences in LRP5 and LRP6 dependency.

The WNT/ß-catenin signalling cascade is the best-investigated frizzled receptor (FZD) pathway, however, whether and how specific combinations of WNT/FZD and co-receptors LRP5 and LRP6 differentially affect this pathway are not well understood. This is mostly due to the fact that there are 19 WNTs, 10 FZDs and at least two co-receptors. In our attempt to identify the signalling capabilities of specific WNT/FZD/LRP combinations we made use of our previously reported TCF/LEF Gaussia luciferase reporter gene HEK293 cell line (Ring et al., 2011). Generation of WNT/FZD fusion constructs - but not their separate transfection - without or with additional isogenic overexpression of LRP5 and LRP6 in our reporter cells permitted the investigation of specific WNT/FZD/LRP combinations. The canonical WNT3a in fusion to almost all FZDs was able to induce ß-catenin-dependent signalling with strong dependency on LRP6 but not LRP5. Interestingly, noncanonical WNT ligands, WNT4 and WNT5a, were also able to act "canonically" but only in fusion with specific FZDs and with selective dependence on LRP5 or LRP6. These data and extension of this experimental setup to the poorly characterized other WNTs should facilitate deeper insight into the complex WNT/FZD signalling system and its function.

miR-92a regulates TGF-ß1-induced WISP1 expression in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and fatal form of idiopathic interstitial pneumonia. MicroRNAs (miRNAs), short, single-stranded RNAs that regulate protein expression in a post-transcriptional manner, have recently been demonstrated to contribute to IPF pathogenesis. We have previously identified WNT1-inducible signaling pathway protein 1 (WISP1) as a highly expressed pro-fibrotic mediator in IPF, but the underlying mechanisms resulting in increased WISP1 expression, remain elusive. Here, we investigated whether WISP1 is a target of miRNA regulation. We applied a novel supervised machine learning approach, which predicted miR-30a/d and miR-92a target sites in regions of the human WISP1 3'UTR preferentially bound by the miRNA ribonucleoprotein complex. Both miRNAs were decreased in IPF samples, whereas WISP1 protein was increased. We demonstrated further that transforming growth factor (TGF)-ß1-induced WISP1 expression in primary lung fibroblasts in vitro and lung homogenates in vivo. Notably, miR-30a and miR-92a reversed TGF-ß1-induced WISP1 mRNA expression in lung fibroblasts. Moreover, miR-92a inhibition increased WISP1 protein expression in lung fibroblasts. An inverse relationship for WISP1 and miR-92a was found in a TGF-ß1 dependent lung fibrosis model in vivo. Finally, we found significantly increased WISP1 expression in primary IPF fibroblasts, which negatively correlated with miR-92a level ex vivo. Altogether, our findings indicate a regulatory role of miR-92a for WISP1 expression in pulmonary fibrosis.

MicroRNAs in flow-dependent vascular remodelling.

Changes in haemodynamic forces in the vascular system result in an altered expression of miRs, which play important gene-regulatory roles by pairing to the mRNAs of protein-coding genes to fine-tune post-transcriptional repression. The development and structure of blood vessels are highly adapted to haemodynamic forces, such as shear stress, cyclic stretch, and circumferential wall stress, generated by the conductance of blood. Thus, fluctuations in shear stress contribute to miR-regulated differential gene expression in endothelial cells (ECs), which is essential for maintenance of vascular physiology. Several microRNAs have been identified that are induced by high shear stress mediating an atheroprotective role, such as miR-10a, miR-19a, miR-23b, miR-101, and miR-143/145. While changes in the expression profile of miR-21 and miR-92a by high shear stress are associated with an atheroprotective function, low shear stress-induced expression of miR-21, miR-92a, and miR-663 results in a pathological EC phenotype. MiR-155 fulfils pleiotropic functions in different regions of vasculature, when exposed to different modes of shear stress. Thus, changes in shear stress result in differential expression of numerous miRs, triggering the balance between susceptibility and resistance to cardiovascular diseases. Further elucidating the regulation of miRs by flow may allow future clinical applications of miRs as diagnostic and therapeutic tools.

LRP6 mediates Wnt/ß-catenin signaling and regulates adipogenic differentiation in human mesenchymal stem cells.

Human mesenchymal stem cells (hMSC) are subjected to the control of several signal transduction pathways during regeneration processes, whereby Wnt/ß-catenin signaling is of pivotal importance. Since there exists only fragmentary knowledge concerning the molecular function of the Wnt-coreceptors LRP5 and LRP6 (low-density lipoprotein receptor-related protein) in hMSC, we studied their impact on Wnt/ß-catenin signal transduction by RNA interference. For monitoring changes in ß-catenin-dependent transcription in a highly sensitive and specific manner, hMSC were stably transfected with a TCF/LEF reporter gene plasmid. In the presence of the activator Wnt3a, knockdown of LRP6 led to a strong decreased Wnt/ß-catenin signaling, while RNAi against LRP5 exhibited no effect in this setting. In a reverse approach, ectopic expression of LRP6 resulted in a strong enhancement of Wnt/ß-catenin signaling, whereas overexpression of LRP5 exhibited no increased signaling capacity. Furthermore, only the ectopic expression of LRP6--but not that of LRP5--was able to restore Wnt3a-mediated ß-catenin signaling after knockdown of endogenously expressed LRP6. These results demonstrate LRP6 as the predominant Wnt3a LRP-receptor in hMSC, which cannot be substituted by LRP5. In addition, we observed enhanced differentiation toward the adipogenic lineage after RNAi against LRP6 which was associated with the induction of PPAR-γ and fat vacuole formation. Thus, LRP6 is not only indispensable for Wnt3a/ß-catenin signaling, but also for the suppression of differentiation of hMSC into the adipogenic lineage. Based on these observations, LRP6 may represent an attractive drug target for manipulating hMSC in cell and tissue regeneration approaches.

Human osteoblast-derived factors induce early osteogenic markers in human mesenchymal stem cells.

The capacity of human mesenchymal stem cells (hMSC) for self-renewal and differentiation is a tightly regulated process within their microenvironment--the stem cell niche. For future therapeutic applications of hMSC within the frame of tissue engineering, it is of major importance to understand the factors involved in triggering differentiation cascades of hMSC. Using either osteoblast-conditioned medium or an indirect coculture system, we investigated whether soluble factors from human osteoblasts (hOB) are sufficient to induce early osteogenic markers in hMSC. Thereby, we detected an induction of several osteogenic markers like alkaline phosphatase, bone sialoprotein 2, leptin receptor, decorin, and cathepsin K in hMSC as indicators of the onset of early osteogenesis. Further, because Wnt signaling has been reported to play an important role in osteogenesis, we performed RNAi against the main Wnt mediator beta-catenin and the low-density lipoprotein receptor-related protein 5 as a major Wnt co-receptor in hMSC. Whereas alkaline phosphatase was significantly downregulated with this approach, the other osteogenic markers showed a markedly upregulation. These observations suggest that hOB-secreted factors could induce early osteogenic markers in hMSC. Thus, with regard to a therapeutic setting, these findings may pave the way for a more in vivo-related differentiation procedure for the generation of osteoblast-like cells.

T-SP1: a novel serine protease-like protein predominantly expressed in testis.

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.

Cellular expression of plasma prekallikrein in human tissues.

Plasma prekallikrein (PPK) is synthesised in hepatocytes and secreted into the blood, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Recently we demonstrated by quantitative RT-PCR that the human PPK gene is transcribed not only in the liver, but also in various non-hepatic human tissues at significant levels. However, up to now no reliable information is available concerning protein synthesis in the corresponding human tissues. Here we demonstrate by immunohistochemical studies that PPK or plasma kallikrein (PK) is localised in cells of different embryologically derived human tissues. In the human nephron, single cells of the distal tubules stained intensely, while the cytoplasm of cells forming proximal tubules and collecting ducts stained uniformly. PPK/PK was localised in hepatic epithelial cells of the liver, in cells of the pancreatic islet of Langerhans, in the interstitial Leydig cells of the testes, in the follicular and thecal granulosa cells of the ovary, and in the parotid gland, oesophagus, skin, respiratory tract, prostate and breast. We conclude that the cellular localisation of PPK/PK in multiple different progenitor-derived cells indicates specific cellular functions of this enzyme, in addition to its known function in the blood.

MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines.

Human mesenchymal stem cells (hMSCs) represent promising tools in various clinical applications, including the regeneration of injured tissues by endogenous or transplanted hMSCs. The molecular mechanisms, however, that control hMSC mobilization and homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. We have analyzed bone marrow-derivedhMSCs and detected strong expression and synthesis of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. The ability of hMSCs to traverse reconstituted human basement membranes was effectively blocked in the presence of synthetic MMP inhibitors. Detailed studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired hMSC invasion, whereas silencing of TIMP-1 enhanced cell migration, indicating opposing roles of both TIMPs in this process. Moreover, the inflammatory cytokines TGF-beta1, IL-1beta, and TNF-alpha up-regulated MMP-2, MT1-MMP, and/or MMP-9 production in these cells, resulting in a strong stimulation of chemotactic migration through ECM, whereas the chemokine SDF-1alpha exhibited minor effects on MMP/TIMP expression and cell invasion. Thus, induction of specific MMP activity in hMSCs by inflammatory cytokines promotes directed cell migration across reconstituted basement membranes in vitro providing a potential mechanism in hMSC recruitment and extravasation into injured tissues in vivo.

Human mesenchymal stem cells at the single-cell level: simultaneous seven-colour immunofluorescence.

Extracellular, intracellular or surface proteins can be used as putative markers to characterize human mesenchymal stem cells (hMSC). However, these markers are also expressed by other cell types and primary cell pools reveal considerable heterogeneity. Therefore, the simultaneous detection of several markers on a single cell appears to be an attractive approach to identify hMSC. Here we demonstrate the specific distinction of human MSC from human osteoblasts via seven-colour fluorescence on the single cell level with simultaneous marker detection of CD44, CD105/endoglin, CD106/VCAM-1, collagen-IV, fibronectin, actin and DAPI nuclear staining. We performed spectral image acquisition using a Sagnac-type interferometer. Subsequent linear unmixing allowed for decomposition of each pixel in its spectral components. Our approach reveals a typical expression profile of the adherent singular cells, allowing the specific distinction between hMSC and osteoblasts on the single cell level.