Juan F. Santibanez

TGF-β/TGF-β receptor system and its role in physiological and pathological conditions.

The TGF-β (transforming growth factor-β) system signals via protein kinase receptors and Smad mediators to regulate a plethora of biological processes, including morphogenesis, embryonic development, adult stem cell differentiation, immune regulation, wound healing and inflammation. In addition, alterations of specific components of the TGF-β signalling pathway may contribute to a broad range of pathologies such as cancer, cardiovascular pathology, fibrosis and congenital diseases. The knowledge about the mechanisms involved in TGF-β signal transduction has allowed a better understanding of the disease pathogenicity as well as the identification of several molecular targets with great potential in therapeutic interventions.

ROS-NFκΒ mediates TGF-β1-induced expression of urokinase-type plasminogen activator, matrix metalloproteinase-9 and cell invasion

TGF-β1 has been postulated as a pro-oncogenic factor in the late step of the tumoral progression. In transformed cells, TGF-β1 enhances the capacity to degrade the extracellular matrix, cell invasiveness and epithelial-mesenchymal transition, which are crucial steps for metastasis. Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) are critical components in cell migration and invasion induced by TGF-β1, however, the exact mechanism by which TGF-β1 regulates uPA and MMP-9 is not well elucidated so far. In the present study, we analyzed the role of ROS-NFκΒ, signal as mediator in the cell malignity enhancement by TGF-β1. We found that TGF-β1 activates NFκΒ, through Rac1-NOXs-ROS-dependent mechanism. Our results shows that TGF-β1 stimulation of uPA and MMP-9 expression involve NOXs-dependent ROS and NFκΒ, activation, demonstrated by using DPI, NOXs inhibitor, ROS scavenger N-acetylcysteine and SN50, an NFkb inhibitor. Furthermore, we found that the inhibition of ROS and NFκΒ, abrogates TGF-β1 stimulation of EMT, cell motility and invasion. Thus, ROS-NFκΒ acts as the crucial signal in TGF-β1-induced uPA and MMP-9 expression thereby mediating the enhancement of cellular malignity by TGF-β1.

Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2

The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment.

SMAD3 is essential for transforming growth factor-β1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes.

Transforming growth factor-β1 (TGF-β1) stimulates the extracellular matrix degrading proteases expression and cell migration in order to enhance cancer cells malignancy. In the present study, we analysed the role of TGF-β1-induced Smad3 activation in the urokinase type plasminogen activator (uPA) production, as well as in cell migration and E-cadherin downregulation in transformed PDV keratinocyte cell line. TGF-β1 signalling was interfered by the chemical inhibitor of the TGF-β1-receptor 1 (ALK5), SB505124, and the specific Smad3 inhibitor, SiS3. Our results showed that TGF-β1 stimulates uPA expression directly through ALK5 activation. The inhibition of Smad3 strongly reduced the capacity of TGF-β1 to stimulate uPA expression, in parallel decreasing the uPA inhibitor plasminogen activator inhibitor type 1 (PAI-1) expression. In addition, the transient expression of dominant negative Smad3 mutant inhibited the TGF-β1-induced uPA promoter transactivation. Moreover, Smad3-/- mouse embryonic fibroblasts were refractory to the induction of uPA by TGF-β1. The inhibition of both ALK5 and Smad3 dramatically blocked the TGF-β1-stimulated E-cadherin downregulation, F-actin reorganisation and migration of PDV cells. Taken together, our results suggest that the TGF-β1-induced activation of Smad3 is the critical step for the uPA upregulation and E-cadherin downregulation, which are the key events preceding the induction of cell migration by TGF-β1 in transformed cells.

The TGF-β co-receptor endoglin modulates the expression and transforming potential of H-Ras

Endoglin is a coreceptor for transforming growth factor-β (TGF-β) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-β1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-β1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-β type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-β1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis.

Urokinase type plasminogen activator mediates Interleukin-17-induced peripheral blood mesenchymal stem cell motility and transendothelial migration

Mesenchymal stem cells (MSCs) have the potential to migrate toward damaged tissues increasing tissue regeneration. Interleukin-17 (IL-17) is a proinflammatory cytokine with pleiotropic effects associated with many inflammatory diseases. Although IL-17 can modulate MSC functions, its capacity to regulate MSC migration is not well elucidated so far. Here, we studied the role of IL-17 on peripheral blood (PB) derived MSC migration and transmigration across endothelial cells. IL-17 increased PB-MSC migration in a wound healing assay as well as cell mobilization from collagen gel. Concomitantly IL-17 induced the expression of urokinase type plasminogen activator (uPA) without affecting matrix metalloproteinase expression. The incremented uPA expression mediated the capacity of IL-17 to enhance PB-MSC migration in a ERK1,2 MAPK dependent way. Also, IL-17 induced PB-MSC migration alongside with changes in cell polarization and uPA localization in cell protrusions. Moreover, IL-17 increased PB-MSC adhesion to endothelial cells and transendothelial migration, as well as increased the capacity of PB-MSC adhesion to fibronectin, in an uPA-dependent fashion. Therefore, our data suggested that IL-17 may act as chemotropic factor for PB-MSCs by incrementing cell motility and uPA expression during inflammation development.

IL-17 and FGF signaling involved in mouse mesenchymal stem cell proliferation.

The mouse is a suitable experimental model to study the biology of mesenchymal stem cells (MSCs), as well as to be used in biocompatibility studies and tissue engineering models. However, the isolation and purification of murine MSCs is far more challenging than their counterparts from other species. In this study, we isolated, expanded and characterized mouse MSCs from bone marrow (BM-MSCs). Additionally, we analyzed the effects of two regulatory molecules, interleukin 17 (IL-17) and basic fibroblast growth factor (bFGF), on BM-MSCs growth and elucidated the signaling pathways involved. The results revealed that IL-17 increased the frequency of colony-forming units fibroblast (CFU-F) as well as the BM-MSCs proliferation in a dose-dependent manner, while bFGF supplementation had no significant effect on CFU-F frequency but induced an increase in cell proliferation. Their combined usage did not produce additive effects on BM-MSCs proliferation and even induced reduction in the number of CFU-F. Also, the involvement of both p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) signaling in proliferative activity of IL-17 and bFGF on murine BM-MSCs and, moreover, the increased co-activation of a common signaling molecule, p38 MAPK, were demonstrated. Together, the data presented highlighted the role of IL-17 and bFGF in murine BM-MSCs proliferation and pointed to the complexity and specificity of the signaling networks leading to MSCs proliferation in response to different regulatory molecules.

Interleukin-17 modulates myoblast cell migration by inhibiting urokinase type plasminogen activator expression through p38 mitogen-activated protein kinase.

Interleukin-17 belongs to a family of pro-inflammatory cytokines with pleiotropic effects, which can be associated with several inflammatory diseases of the muscle tissue. Although elevated levels of interleukin-17 have been described in inflammatory myopathies, its role in muscle homeostasis remains to be elucidated. The requirement of the urokinase type plasminogen activator in skeletal myogenesis was recently demonstrated in vivo and in vitro, suggesting its involvement in the regulation of extracellular matrix remodeling, cell migration and myoblast fusion. Our previous results have demonstrated that interleukin-17 inhibits myogenic differentiation of C2C12 myoblasts in vitro concomitantly with the inhibition of cell migration. However, the involvement of urokinase type plasminogen activator in interleukin-17-inhibited myogenesis and migration remained to be analyzed. Therefore, the effect of interleukin-17 on the production of urokinase type plasminogen activator by C2C12 myoblasts was determined in the present study. Our results demonstrated that interleukin-17 strongly inhibits urokinase type plasminogen activator expression during myogenic differentiation. This reduction of urokinase type plasminogen activator production corresponded with the inhibition of cell migration by interleukin-17. Activation of p38 signaling pathway elicited by interleukin-17 mediated the inhibition of both urokinase type plasminogen activator expression and cell migration. Additionally, IL-17 inhibited C2C12 cells migration by causing the cells to reorganize their cytoskeleton and lose polarity. Therefore, our results suggest a novel mechanism by which interleukin-17 regulates myogenic differentiation through the inhibition of urokinase type plasminogen activator expression and cell migration. Accordingly, interleukin-17 may represent a potential clinical target worth investigating for the treatment of inflammatory muscle diseases.

SKIP is required for TGF-β1-induced epithelial mesenchymal transition and migration in transformed keratinocytes

Transforming growth factor‐β1 (TGF‐β1) potently induces the epithelial‐mesenchymal transition (EMT) during tumoral progression. Although Sky‐interacting protein (SKIP) regulates TGF‐β1‐induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF‐β1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS‐S, Smad3 activation decreased, along with an inhibition of TGF‐β1‐induced EMT, and the cells were sensitized to the TGF‐β1‐dependent inhibition of proliferation. Also, AS‐S cells showed a weaker migration and invasion response. Moreover, TGF‐β1‐induced urokinase‐type plasminogen activator expression was inhibited, concomitantly with a TGF‐β1‐independent increment of the plasminogen‐activator inhibitor‐1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF‐β1 in transformed cells.

Transforming growth factor-β superfamily, implications in development and differentiation of stem cells

Abstract Transforming growth factor-β (TGF-β) family members, including TGF-βs and bone morphogenetic proteins (BMPs), play important roles in directing the fate of stem cells. In embryonic stem cells, the TGF-β superfamily participates in almost all stages of cell development, such as cell maintenance, lineage selection, and progression of differentiation. In adult mesenchymal stem cells (MSCs), TGF-βs can provide competence for early stages of chondroblastic and osteoblastic differentiation, but they inhibit myogenesis, adipogenesis, and late-stage osteoblast differentiation. BMPs also inhibit adipogenesis and myogenesis, but they strongly promote osteoblast differentiation. The TGF-β superfamily members signal via specific serine/threonine kinase receptors and their nuclear effectors termed Smad proteins as well as through non-Smad pathways, which explain their pleiotropic effects in self-renewal and differentiation of stem cells. This review summarizes the current knowledge on the pleiotropic effects of the TGF-β superfamily of growth factors on the fate of stem cells and also discusses the mechanisms by which the TGF-β superfamily members control embryonic and MSCs differentiation.