Tumor necrosis factor-α and transforming growth factor-ß1 facilitate differentiation and proliferation of tendon-derived stem cells in vitro.

OBJECTIVES: To investigate the effects of tumor necrosis factor-α (TNF-α) and transforming growth factor-ß1 (TGF-ß1) on the proliferation and differentiation of tendon-derived stem cells (TDSC). RESULTS: TNF-α inhibits the proliferation and tenogenic/osteogenic differentiation of TDSC but, after simultaneous or sequential treatment with TGF-ß1 and TNF-α, the expression of tenogenic/osteogenic-related marker and proliferation of TDSC was significantly increased. During these processes, Smad2/3 and Smad1/5/8 were highly phosphorylated, meaning that the TGF-ß and BMP signaling pathways were highly activated. Further study revealed that the expression of Inhibitor-Smad appeared to be negatively correlated to the proliferation and differentiation of TDSC. CONCLUSIONS: Combining the use of TNF-α and TGF-ß1 could improve the proliferation and differentiation of TDSC in vitro, and the expression of I-Smad is negatively correlated with TDSC proliferation and differentiation.

[Effects of blocking two sites of transforming growth factor-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts].

OBJECTIVE: To explore the effects of blocking two sites of TGF-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts. METHODS: Two lentivirus vectors encoding soluble TGF-ß receptor II (sTßRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTßRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-ß1 for 72 h; sTßRII group, transfected with lenti-sTßRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-ß1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test). RESULTS: (1) HFF-1 cells transfected with lenti-sTßRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTßRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTßRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTßRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05). CONCLUSIONS: In human skin fibroblasts, blockage of two sites of TGF-ß/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-ß1 to a greater extent than that of blocking one single site.

Neural commitment of human pluripotent stem cells under defined conditions recapitulates neural development and generates patient-specific neural cells.

Standardization of culture methods for human pluripotent stem cell (PSC) neural differentiation can greatly contribute to the development of novel clinical advancements through the comprehension of neurodevelopmental diseases. Here, we report an approach that reproduces neural commitment from human induced pluripotent stem cells using dual-SMAD inhibition under defined conditions in a vitronectin-based monolayer system. By employing this method it was possible to obtain neurons derived from both control and Rett syndrome patients' pluripotent cells. During differentiation mutated cells displayed alterations in the number of neuronal projections, and production of Tuj1 and MAP2-positive neurons. Although investigation of a broader number of patients would be required, these observations are in accordance with previous studies showing impaired differentiation of these cells. Consequently, our experimental methodology was proved useful not only for the generation of neural cells, but also made possible to compare neural differentiation behavior of different cell lines under defined culture conditions. This study thus expects to contribute with an optimized approach to study the neural commitment of human PSCs, and to produce patient-specific neural cells that can be used to gain a better understanding of disease mechanisms.

Inhibitory SMADs: potential regulators of ovarian function.

Transforming growth factor beta (TGFB) superfamily signaling regulates essential reproductive functions. Dysregulation of TGFB signaling results in cellular and molecular deficiencies in the ovary, leading to reproductive diseases and cancer development. SMAD proteins are canonical TGFB signaling components consisting of receptor-regulated SMADs (SMAD1/2/3/5/9), a common SMAD (SMAD4), and inhibitory SMADs (SMAD6/7). Inhibitory SMADs are negative regulators of TGFB and bone morphogenetic protein signaling, and their reproductive functions are poorly defined. Emerging evidence supports that inhibitory SMADs are potential regulators of ovarian function. Further efforts and new genetic models are needed to unveil the role of inhibitory SMADs in the ovary.

Effects of blocking two sites of transforming growth factor-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of blocking two sites of TGF-β/Smads signaling on the formation of scar-related proteins in human skin fibroblasts.</p><p><b>METHODS</b>Two lentivirus vectors encoding soluble TGF-β receptor II (sTβRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTβRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-β1 for 72 h; sTβRII group, transfected with lenti-sTβRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-β1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test).</p><p><b>RESULTS</b>(1) HFF-1 cells transfected with lenti-sTβRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTβRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTβRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTβRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05).</p><p><b>CONCLUSIONS</b>In human skin fibroblasts, blockage of two sites of TGF-β/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-β1 to a greater extent than that of blocking one single site.</p>

Ter94 ATPase complex targets k11-linked ubiquitinated ci to proteasomes for partial degradation.

The Cubitus interruptus (Ci)/Gli family of transcription factors can be degraded either completely or partially from a full-length form (Ci155/Gli(FL)) to a truncated repressor (Ci75/Gli(R)) by proteasomes to mediate Hedgehog (Hh) signaling. The mechanism by which proteasomes distinguish ubiquitinated Ci/Gli to carry out complete versus partial degradation is not known. Here, we show that Ter94 ATPase and its mammalian counterpart, p97, are involved in processing Ci and Gli3 into Ci75 and Gli3(R), respectively. Ter94 regulates the partial degradation of ubiquitinated Ci by Cul1-Slimb-based E3 ligase through its adaptors Ufd1-like and dNpl4. We demonstrate that Cul1-Slimb-based E3 ligase, but not Cul3-Rdx-based E3 ligase, modifies Ci by efficient addition of K11-linked ubiquitin chains. Ter94(Ufd1-like/dNpl4) complex interacts directly with Cul1-Slimb, and, intriguingly, it prefers K11-linked ubiquitinated Ci. Thus, Ter94 ATPase and K11-linked ubiquitination in Ci contribute to the selectivity by proteasomes for partial degradation.

Differences in activation of mouse hepcidin by dietary iron and parenterally administered iron dextran: compartmentalization is critical for iron sensing.

The iron regulatory hormone hepcidin responds to both oral and parenteral iron. Here, we hypothesized that the diverse iron trafficking routes may affect the dynamics and kinetics of the hepcidin activation pathway. To address this, C57BL/6 mice were administered an iron-enriched diet or injected i.p. with iron dextran and analyzed over time. After 1 week of dietary loading with carbonyl iron, mice exhibited significant increases in serum iron and transferrin saturation, as well as in hepatic iron, Smad1/5/8 phosphorylation and bone morphogenetic protein 6 (BMP6), and hepcidin mRNAs. Nevertheless, hepcidin expression reached a plateau afterward, possibly due to upregulation of inhibitory Smad7, Id1, and matriptase-2 mRNAs, while hepatic and splenic iron continued to accumulate over 9 weeks. One day following parenteral administration of iron dextran, mice manifested elevated serum and hepatic iron levels and Smad1/5/8 phosphorylation, but no increases in transferrin saturation or BMP6 mRNA. Surprisingly, hepcidin failed to appropriately respond to acute overload with iron dextran, and a delayed (after 5-7 days) hepcidin upregulation correlated with increased transferrin saturation, partial relocation of iron from macrophages to hepatocytes, and induction of BMP6 mRNA. Our data suggest that the physiological hepcidin response is saturable and are consistent with the idea that hepcidin senses exclusively iron compartmentalized within circulating transferrin and/or hepatocytes.

Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration.

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.

TGF-beta-dependent and -independent roles of STRAP in cancer.

The serine-threonine kinase receptor-associated protein (STRAP) was initially identified as a putative inhibitor of the canonical TGF-beta signaling pathway. Because the Smad-dependent TGF-beta pathway negatively regulates cellular growth, early functional studies suggested that STRAP behaves as an oncogene. Indeed, a correlation between STRAP overexpression and various cancers has been identified. With the emergence of new studies on the biological function of STRAP, it is becoming clear that STRAP regulates several distinct cellular processes and modulates multiple signaling pathways. While STRAP itself does not possess enzymatic activity, it appears that STRAP influences biological processes through associations with cellular proteins. In this review, we will describe the TGF-beta-dependent and -independent functions of STRAP and provide a context for the significance of STRAP activity in the development of cancer.

TGF-beta signaling and the role of inhibitory Smads in non-small cell lung cancer.

The signaling pathway mediated by transforming growth factor-beta (TGF-beta) participates in various biologic processes, including cell growth, differentiation, angiogenesis, apoptosis, and extracellular matrix remodeling. In the context of cancer, TGF-beta signaling can inhibit tumor growth in early-stage tumors. However, in late-stage tumors, the very same pathway promotes tumor invasiveness and metastasis. This paradoxical effect is mediated through similar to mothers against decapentaplegic or Smad protein dependent and independent mechanisms and provides an opportunity for targeted cancer therapy. This review summarizes the molecular process of TGF-beta signaling and the changes in inhibitory Smads that contribute to lung cancer progression. We also present current approaches for rational therapies that target the TGF-beta signaling pathway in cancer.

Modeling and analysis of MH1 domain of Smads and their interaction with promoter DNA sequence motif.

The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta (TGF-beta) superfamily of proteins at the cell surface. The prototypic members of the Smad family, Mad and Sma, were first described in Drosophila and Caenorhabditis elegans, respectively. Related proteins in Xenopus, Humans, Mice and Rats were subsequently identified, and are now known as Smads. Smad protein family members act downstream in the TGF-beta signaling pathway mediating various biological processes, including cell growth, differentiation, matrix production, apoptosis and development. Smads range from about 400-500 amino acids in length and are grouped into the receptor-regulated Smads (R-Smads), the common Smads (Co-Smads) and the inhibitory Smads (I-Smads). There are eight Smads in mammals, Smad1/5/8 (bone morphogenetic protein regulated) and Smad2/3 (TGF-beta/activin regulated) are termed R-Smads, Smad4 is denoted as Co-Smad and Smad6/7 are inhibitory Smads. A typical Smad consists of a conserved N-terminal Mad Homology 1 (MH1) domain and a C-terminal Mad Homology 2 (MH2) domain connected by a proline rich linker. The MH1 domain plays key role in DNA recognition and also facilitates the binding of Smad4 to the phosphorylated C-terminus of R-Smads to form activated complex. The MH2 domain exhibits transcriptional activation properties. In order to understand the structural basis of interaction of various Smads with their target proteins and the promoter DNA, we modeled MH1 domain of the remaining mammalian Smads based on known crystal structures of Smad3-MH1 domain bound to GTCT Smad box DNA sequence (1OZJ). We generated a B-DNA structure using average base-pair parameters of Twist, Tilt, Roll and base Slide angles. We then modeled interaction pose of the MH1 domain of Smad1/5/8 to their corresponding DNA sequence motif GCCG. These models provide the structural basis towards understanding functional similarities and differences among various Smads.

Regulating the stability of TGFbeta receptors and Smads.

Transforming growth factor beta (TGFbeta) controls cellular behavior in embryonic and adult tissues. TGFbeta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that together regulate gene expression. In this review, mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of TGFbeta are presented. We discuss how the activity and duration of TGFbeta receptor/Smad signaling can be regulated by post-translational modifications that affect the stability of key proteins in the pathway. We highlight links between these mechanisms and human diseases, such as tissue fibrosis and cancer.

Bone morphogenetic protein 2 therapy for craniofacial surgery.

Between 2 and 10 years of age, the developing craniofacial skeleton poses a significant reconstructive challenge. Local autogenous bone is largely unavailable, distant bone grafts are fraught with significant morbidity and limited yield, and alloplastic materials are incompatible with the growing calvarium and facial skeleton. Bone morphogenetic protein (BMP) 2, a member of a class of proteins first noticed in the 1960s to promote bone deposition in soft tissues, offers a potential solution to the bone shortage historically faced by the pediatric craniofacial surgeon. A review of English language literature was conducted from the 1960s to the present.Attention was focused on BMP-2's osteoinductive mechanism, basic science and translational laboratory findings, and multidisciplinary clinical experiences. Bone morphogenetic protein 2 has been embraced by spine surgeons, is gaining in popularity for long-bone repair, and is making its way into the plastic surgery literature. Bone morphogenetic protein 2 may provide a basis for an off-the-shelf tissue-engineered bone construct that is compatible with the growing craniofacial skeleton while free from the morbidities of distant graft harvest. Questions remain, however, regarding the safety and efficacy of this compound in the context of pediatric craniofacial surgery. In an effort to facilitate the clinician's risk-benefit analysis of this emerging technology, we present a primer on the basic science of BMP-2, a discussion of possible morbidities associated with its use, a review of laboratory and clinical trials with this substance to date, and an analysis of strategies to maximize its efficacy in craniofacial surgery.

Structure-function relationship of inhibitory Smads: Structural flexibility contributes to functional divergence.

Smads are a small family of eukaryotic transcription regulators that play key roles in the transforming growth factor-beta signaling cascade. Smad6 and Smad7, the inhibitory or I-Smads, inhibit signaling downstream of TGF-beta type I receptors, thereby acting as negative regulators of signaling mediated by TGF-beta superfamily of ligands. Smad6 is known to specifically inhibit BMP type I receptor mediated signaling, while Smad7 is a more general inhibitor, able to block signaling mediated by a set of related TGF-beta type I receptors, including type I receptors for BMP and TGF-beta/Activin. In this study we have sought to understand the structural basis for this functional divergence of I-Smads. We have created homology-based models for the MH1 and MH2 domains of the two I-Smads and have carried out detailed molecular dynamics (MD) simulations of these proteins in explicit solvent to investigate the flexibility of the domains. The molecular models show that the I-Smads have lost many of the secondary structural elements found in the R-Smads, giving rise to longer loops in the tertiary structure of Smad6 and Smad7. Detailed analysis of the structural models and the MD trajectories clearly reveal that compared to Smad6, Smad7 has a more flexible overall folding, marked by the presence of highly flexible amino acid residues in functionally important regions of the protein. Interestingly, three of these residues-Phe411, Lys401, and Cys406, map to L3 loop of Smad7 MH2 domain, which is a critical structural determinant in Smad-type I receptor interactions. The increased structural flexibility of Smad7, arising out of longer, more flexible loops in its MH2 domain, might enable Smad7 to interact with a set of related yet structurally diverse type I receptors. Taken together with experimental evidence published in recent literature that hint at structural factors underlying the generic nature of inhibition by Smad7, our results strongly suggest that structural flexibility could be a prime contributor to the functional differences between Smad6 and Smad7. Additionally, we have been able to use the Smad7 structural model to successfully rationalize the results of in vitro site-specific mutagenesis experiments in published literature. This also provides biological validation for our model. Apart from this, analysis of the MH1 molcular model of Smad6 delineates a basic patch on the surface of the domain that might take part in nonspecific DNA binding by Smad6. This finding is consistent with earlier experimental data and is relevant since the characteristic beta-hairpin DNA binding element of R-Smads is completely absent in the I-Smads. Finally, the molecular models described here can serve to guide future biochemical and genetic studies on I-Smads.

Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts.

Transforming growth factor-beta(1) (TGF-beta(1)) signal and downstream Smads play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 (I-Smad7) is an inducible regulatory Smad protein that antagonizes TGF-beta(1) signal mediated via direct abrogation of R-Smad phosphorylation. The effect of ectopic I-Smad7 on net collagen production was investigated using hydroxyproline assay. Adenovirus-mediated I-Smad7 gene (at 100 multiplicity of infection) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-beta(1) in primary rat cardiac myofibroblasts. In I-Smad7-infected cells, we also observed the ablation of TGF-beta(1)-induced R-Smad2 phosphorylation vs. LacZ controls. Overdriven I-Smad7 was associated with significantly increased expression of immunoreactive 65-kDa matrix metalloproteinase-2 (MMP-2) protein in culture medium of myofibroblast compared with LacZ-infected cells. Expression of the 72-kDa MMP-2 variant, e.g., the inactive form, was not altered by exogenous I-Smad7 transfection/overexpression. Furthermore, I-Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced [(3)H]thymidine incorporation vs. Ad-LacZ-infected controls. We suggest that negative modulation of R-Smad phosphorylation by ectopic I-Smad7 may contribute to the downregulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of I-Smad7 may provide a new therapeutic strategy for cardiac fibrosis.

Crk-associated substrate lymphocyte type regulates transforming growth factor-beta signaling by inhibiting Smad6 and Smad7.

Crk-associated substrate lymphocyte type (Cas-L) is a 105 kDa docking protein with diverse functional properties, including regulation of cell division, proliferation, migration and adhesion. Cas-L is also involved in beta1 integrin- or antigen receptor-mediated signaling in B and T cells. In the present study, we demonstrate that Cas-L potentiates transforming growth factor-beta (TGF-beta) signaling pathway by interacting with Smad6 and Smad7. Immunoprecipitation experiments reveal that single domain deletion of full-length Cas-L completely abolishes its docking function with Smad6 and Smad7, suggesting that the natural structure of Cas-L is necessary for its association with Smad6 and Smad7. On the other hand, both N-terminal and C-terminal deletion mutants of Smad6 and Smad7 still retain their docking ability to Cas-L, suggesting that Smad6 and Smad7 possess several binding motifs to Cas-L. Moreover, Cas-L interaction with Mad-homology (MH)2 domain, but not with MH1 domain of Smad6 or Smad7, ameliorates TGF-beta-induced signaling pathway. Finally, depletion of Cas-L by small-interfering RNA oligo attenuates TGF-beta-induced growth inhibition of Huh-7 cells, with a concomitant reduction in phosphorylation of Smad2 and Smad3. These results strongly suggest that Cas-L is a potential regulator of TGF-beta signaling pathway.

Imbalance of receptor-regulated and inhibitory Smads in lung fibroblasts from bleomycin-exposed rats.

Transforming growth factor (TGF)-beta plays a central role in lung fibrosis, stimulating extracellular matrix deposition. Intracellular signaling of TGF-beta is mediated by Smad proteins. We questioned whether the expression and activation of Smads would be altered in lung fibroblasts from rats exposed to bleomycin, an agent used to provoke an experimental model of lung fibrosis. Fibroblasts were isolated from rat lungs 14 d after intratracheal instillation of bleomycin (BLF) or saline (NLF), and cell cultures established. Whole cell lysates were obtained at baseline, and after stimulation with TGF-beta1 (10 ng/ml). Western blot analysis was performed to measure levels of phosphorylated Smad3 (p-Smad3) and Smad7. Real-time PCR was used to determine changes in Smad7 mRNA after TGF-beta stimulation. We found increased baseline levels of p-Smad3 in BLF versus NLF (P < 0.05). In contrast, baseline levels of Smad7 were comparable. The ratio of stimulatory to inhibitory Smads was increased in BLF compared with NLF (P < 0.05). After stimulation with TGF-beta, levels of p-Smad3 were increased in both groups, with maximal responses at 30 min (P < 0.01). While Smad7 mRNA levels were significantly upregulated (at 1 h) after TGF-beta in both groups, the increase in Smad7 protein was significant in NLF only. We conclude there is sustained activation of Smad signaling in lung fibroblasts isolated from bleomycin-exposed rats, with an imbalance between the levels of p-Smad3 and Smad7. Insufficient levels of the inhibitory Smad7 at baseline, and inadequate response to TGF-beta, may contribute to the fibrotic phenotype characteristic of BLF.

IFN-gamma abrogates profibrogenic TGF-beta signaling in liver by targeting expression of inhibitory and receptor Smads.

BACKGROUND/AIMS: In a randomized open-labeled multicenter trial with patients suffering from chronic HBV infection, we recently identified a benefit of 9-month IFN-gamma treatment resulting in decreased fibrosis scores and a reduced number of alpha-smooth muscle actin-positive hepatic stellate cells (HSCs). Approaches opposing profibrogenic activities of TGF-beta may be amenable in chronic liver disease. According to experimental models, IFN-gamma counteracts several TGF-beta effects. METHODS: The crosstalk of IFN-gamma and TGF-beta signaling relevant for fibrogenesis was investigated in primary cultured rat HSCs and a cell line representing activated HSCs. RESULTS: In vitro studies with HSCs demonstrate that TGF-beta-dependent activation of (CAGA)9-MLP-Luc, a Smad3/4 responsive reporter construct, was significantly decreased by IFN-gamma, indicating a TGF-beta antagonizing function. IFN-gamma induced the activity of the Smad7 promoter and Smad7 protein expression via STAT-1 signaling. In contrast to TGF-beta, IFN-gamma was able to induce Smad7 expression in activated HSCs providing increased protein levels for at least 12h. In addition, expression of Smad2/3 was reduced by IFN-gamma and activation of Smads2/3 was abrogated. CONCLUSIONS: IFN-gamma displays antifibrotic effects in liver cells via STAT-1 phosphorylation, upregulation of Smad7 expression and impaired TGF-beta signaling.

Bone morphogenetic protein activities are enhanced by 3',5'-cyclic adenosine monophosphate through suppression of Smad6 expression in osteoprogenitor cells.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF)-beta superfamily, and some display potent osteogenic activity both in vivo and in vitro. The BMP signaling cascade involving BMP receptors at the cell membrane and intracellular messengers (Smads) has been elucidated, but the regulatory mechanisms of BMP signaling have not been clarified. We previously found that pentoxifyline (PeTx), a nonspecific inhibitor of phosphodiesterase (PDE), and rolipram, a PDE-4-specific inhibitor, enhance BMP-4-induced osteogenic differentiation of mesenchymal cells, probably through the elevation of intracellular cyclic adenosine monophosphate (cAMP) accumulation and modulation of BMP signaling pathways as enhanced BMP-4 action was reproduced by addition of dibutylyl-cAMP (dbcAMP). However, the precise mechanisms underlying the enhancing effects of those agents on BMP signaling were not completely revealed. As already reported, BMPs utilize a specific intracellular signaling cascade to target genes via R-Smads (Smad1,5,8), Co-Smad (Smad4) and I-Smads (Smad6,7). One possibility for cAMP-mediated effects on BMP signaling might be suppression of I-Smads expression since these proteins form a negative feedback loop in BMP signaling. To examine this possibility, changes in I-Smad (Smad6) expression on addition of dbcAMP or PeTx were examined in a bone-marrow-derived osteogenic cell line (ST2). Alkaline phosphatase activity in ST2 cells was consistently induced by BMP-4 treatment (300 ng/ml), and Smad6 mRNA expression was also induced by BMP-4 treatment. Although concurrent treatment of ST2 cells with BMP-4 and dbcAMP elicited further activation of alkaline phosphatase, addition of dbcAMP reduced BMP-4-induced Smad6 expression in a dose-dependent manner. Furthermore, detection of phosphorylated Smad1/5/8 on Western blotting analysis was prolonged, suggesting prolonged kinase activity of BMP receptors through suppressed expression of Smad6. Elevated intracellular cAMP might thus enhance BMP signaling by suppressing Smad6 induction and prolonging intracellular BMP signaling.