The GPI-Anchored Protein Thy-1/CD90 Promotes Wound Healing upon Injury to the Skin by Enhancing Skin Perfusion.

Wound healing is a highly regulated multi-step process that involves a plethora of signals. Blood perfusion is crucial in wound healing and abnormalities in the formation of new blood vessels define the outcome of the wound healing process. Thy-1 has been implicated in angiogenesis and silencing of the Thy-1 gene retards the wound healing process. However, the role of Thy-1 in blood perfusion during wound closure remains unclear. We proposed that Thy-1 regulates vascular perfusion, affecting the healing rate in mouse skin. We analyzed the time of recovery, blood perfusion using Laser Speckle Contrast Imaging, and tissue morphology from images acquired with a Nanozoomer tissue scanner. The latter was assessed in a tissue sample taken with a biopsy punch on several days during the wound healing process. Results obtained with the Thy-1 knockout (Thy-1-/-) mice were compared with control mice. Thy-1-/- mice showed at day seven, a delayed re-epithelialization, increased micro- to macro-circulation ratio, and lower blood perfusion in the wound area. In addition, skin morphology displayed a flatter epidermis, fewer ridges, and almost no stratum granulosum or corneum, while the dermis was thicker, showing more fibroblasts and fewer lymphocytes. Our results suggest a critical role for Thy-1 in wound healing, particularly in vascular dynamics.

High CD90 (THY-1) expression positively correlates with cell transformation and worse prognosis in basal-like breast cancer tumors.

Breast cancer is the most prevalent cancer among women, with the basal-like triple negative (TNBC) being the most agressive one, displaying the poorest prognosis within the ductal carcinoma subtype. Due to the lack of adequate molecular targets, the diagnosis and treatment of patients with the TNBC phenotype has been a great challenge. In a previous work, we identified CD90/Thy-1 as being highly expressed in the aggressive high malignancy grade Hs578T basal-like breast tumor cell line, pointing to this molecule as a promising breast tumor marker, which should be further investigated. Here, CD90 expression was analyzed in human breast cancer samples and its functional role was investigated to better assess the oncogenic nature of CD90 in mammary cells. Quantification of CD90 expression in human breast cancer samples, by tissue microarray, showed that high CD90 positivity correlates with metastasis and poor patient survival in the basal-like subtype. The functional genetic approach, by overexpression in the CD90 cDNA in a basal-like normal mammary cell line (MCF10A) and knockdown in a highly malignant cell line (Hs578T), allowed us to demonstrate that CD90 is involved with several cellular processes that lead to malignant transformation, such as: morphological change, increased cell proliferation, invasiveness, metastasis and activation of the EGFR pathway. Therefore, our results reveal that CD90 is involved with malignant transformation in breast cancer cell lines and is correlated with metastasis and poor patient survival in the basal-like subtype, being considered as a promising new breast cancer target.

Analysis of melanopsin gene expression in the rabbit retina at different ages.

Melanopsin is the photopigment of intrinsically photosensitive retinal ganglion cells that mediate non-visual responses to light. The aim of this study was to describe and analyze melanopsin gene expression in the rabbit retina at different ages and compare its expression with the prototypic gene of retinal ganglion cells (Thy-1 gene). Expression levels of OPN4, Thy-1, and GADPH genes were measured by real-time PCR at 3, 4, 8, 11, 12, 17, 19, 20, 23, 27, 32, and 47 postnatal days. We also regrouped the days before and after day 12 of life (pre-photic and post-photic stage, respectively). Average expression of the OPN4 gene between days was similar (P = 0.713), but was statistically different in the Thy-1 gene (P = 0.004). Also, no significant differences were found in OPN4 gene expression pre-photic and post-photic stage (P = 0.629); however, Thy-1 expression was higher in the pre-photic stage, almost double, than in the post-photic stage, with significant differences (P = 0.001). This is the first report describing OPN4 gene expression in the rabbit retina at different ages. We demonstrated that the OPN4 gene is constantly expressed at all early stages, even before the onset of photoentrainment by the pups and that Thy-1 and OPN4 gene expressions are out of phase.

A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs. METHODS: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors. RESULTS: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression. CONCLUSION: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro.

Wnt/ß-catenin aids in regulating the proliferation of hepG2 cells mediated by thy-1.

Cancer stem cells have been found to play important roles in carcinoma. Although thy-1 has been identified as a potential stem cell marker of liver cancer, whether the Wnt/ß-catenin signaling pathway plays an important role in regulating hepatocarcinoma proliferation and apoptosis mediated by thy-1 remains unknown. Our results showed that high thy-1 expression caused hepG2 cells transfected with a pReceiver-M29/thy-1 eukaryotic expression vector to exhibit obvious heteromorphism, featuring double or multiple nuclei and weaker apoptosis. A high expression of ß-catenin, as a critical signaling protein of Wnt/ß-catenin, and its downstream transcription factor, cyclinD1, were detected in transfected hepG2 cells. We also used aspirin as an inhibitor of the Wnt signaling pathway in the treatment of hepG2 cells transfected with the pReceiver-M29/thy-1 expression vector to make detailed observations of apoptosis in hepG2 cells as well as the differential expression of ß-catenin, cyclinD1, and thy-1. An increasing apoptosis rate was detected in the hepG2 cells and downregulated expression of the three proteins was detected. Hence, we suggest that thy-1 upregulation promotes the proliferation and inhibits apoptosis of hepG2 cells, and that these processes are regulated by the Wnt/ß- catenin signaling pathway.

Differential expression of CD90 and CD14 stem cell markers in malignant breast cancer cell lines.

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells.

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation.

Thy-1, an abundant mammalian glycoprotein, interacts with αvß3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvß3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion.

Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with alphavbeta3 integrin that activates PKCalpha and RhoA.

Clustering of alphavbeta3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC(1) astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCalpha implicated PKCalpha and RhoA activation in these events. Therefore, combined interaction of the astrocyte alphavbeta3-integrin-syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCalpha- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1-integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Developmental regulation of Thy 1.2 rate of synthesis in the mouse cerebellum.

Thy 1.2 is a well-known major cell surface glycoprotein of the central nervous system (CNS). However, the regulation of the expression of this molecule as well as its function are yet to be determined. To approach these problems we studied the synthesis of the molecule in the developing cerebellum of wild-type and staggerer mutant mice. We found the appearance of a [35S]-methionine-labeled band detected with specific Sepharose 4B-bound monoclonal antibodies (Mabs). The Thy 1.2 activity increases progressively from postnatal day 9 (P9), reaching the highest rate at P12, subsequently decreasing sharply at P13, and remaining relatively low up to P16 in the wild type. Comparison of these data to the rates of total protein synthesis reveals a selective developmental regulation of Thy 1.2 expression, at least at the translational level. This correlates quite well with the timing of synaptic stabilization between parallel fibers and Purkinje cell dendritic spines. Furthermore, at P12 Thy 1.2 protein is preferentially located in the synaptosomal fraction. The parallel fiber:Purkinje cell synapsis is not stabilized in the staggerer mutant mouse. At P12 Thy 1.2 synthesis is 30% of the wild type, indicating that the translational regulation of Thy 1.2 is altered in the staggerer mutation.