Local Concentrations of TGF-ß1 and IGF-1 Appear Determinant in Regulating Bone Regeneration in Human Postextraction Tooth Sockets.

Healing after tooth extraction involves a series of reparative processes affecting both alveolar bone and soft tissues. The aim of the present study was to investigate whether activation of molecular signals during the healing process confers a regenerative advantage to the extraction socket soft tissue (ESsT) at 8 weeks of healing. Compared to subepithelial connective tissue graft (CTG), qRT-PCR analyses revealed a dramatic enrichment of the ESsT in osteogenic differentiation markers. However, ESsT and CTG shared characteristics of nonspecialized soft connective tissue by expressing comparable levels of genes encoding abundant extracellular matrix (ECM) proteins. Genes encoding the transforming growth factor-ß1 (TGF-ß1) and its receptors were strongly enriched in the CTG, whereas the transcript for the insulin-like growth factor-1 (IGF-1) showed significantly high and comparable expression in both tissues. Mechanical stimulation, by the means of cyclic strain or matrix stiffness applied to primary ESsT cells (ESsT-C) and CTG fibroblasts (CTG-F) extracted from the tissue samples, revealed that stress-induced TGF-ß1 not exceeding 2.3 ng/mL, as measured by ELISA, in combination with IGF-1 up to 2.5 ng/mL was able to induce the osteogenic potential of ESsT-Cs. However, stiff matrices (50 kPa), upregulating the TGF-ß1 expression up to 6.6 ng/mL, caused downregulation of osteogenic gene expression in the ESsT-Cs. In CTG-Fs, endogenous or stress-induced TGF-ß1 ≥ 4.6 ng/mL was likely responsible for the complete lack of osteogenesis. Treatment of ESsT-Cs with TGF-ß1 and IGF-1 proved that, at specific concentrations, the two growth factors exhibited either an inductive-synergistic or a suppressive activity, thus determining the osteogenic and mineralization potential of ESsT-Cs. Taken together, our data strongly warrant the clinical exploration of ESsT as a graft in augmentative procedures during dental implant placement surgeries.

Cellular responses to deproteinized bovine bone mineral biofunctionalized with bone-conditioned medium.

OBJECTIVES: The aim of the study was to investigate whether the osteoinductive properties of bone-conditioned medium (BCM) harvested from cortical bone chips within a clinically relevant short-term period can enhance the biologic characteristics of deproteinized bovine bone mineral (DBBM) in vitro. MATERIALS AND METHODS: To assess the biofunctionalization of DBBM, the adhesive, proliferative, and differentiation properties of mesenchymal stromal ST2, pre-osteoblastic MC3T3-E1, and primary bone-derived cells grown on BCM-coated DBBM were examined by crystal violet staining of adherent cells, BrdU ELISA, and qRT-PCR, respectively. RESULTS: BCM extracted within 20 min or 24 h in either Ringer's solution (BCM-RS) or RS mixed with autologous serum (BCM-RS + S) increased the adhesive properties of all three cell types seeded on DBBM. The 20-min BCM-RS preparation appeared more potent than the 24-h preparation. BCM-RS made within 20 min or 24 h had strong pro-proliferative effects on all cell types grown on DBBM. RS + S alone exhibited a considerable pro-proliferative effect, suggesting an impact of the serum on cellular growth. DBBM coated with BCM-RS or BCM-RS + S, made within 20 min or 24 h each, caused a significant induction of osteogenic differentiation marker expression with a higher potency of the BCM-RS + S. Finally, a strong additive effect of fresh bone chips combined with BCM-coated DBBM on the osteogenic differentiation of the three cell types was observed. CONCLUSIONS: Altogether, the data strongly support the biofunctionalization of DBBM with BCM extracted within a clinically relevant time window of 20 min. CLINICAL RELEVANCE: Pre-activation of non-osteoinductive biomaterials with BCM, prepared from autologous bone chips during a guided bone regeneration (GBR) procedure, bears the potential of an optimal treatment modality for bone defects in daily practice.

A Novel Volume-Stable Collagen Matrix Induces Changes in the Behavior of Primary Human Oral Fibroblasts, Periodontal Ligament, and Endothelial Cells.

The aim of the present study was to investigate the influence of a novel volume-stable collagen matrix (vCM) on early wound healing events including cellular migration and adhesion, protein adsorption and release, and the dynamics of the hemostatic system. For this purpose, we utilized transwell migration and crystal violet adhesion assays, ELISAs for quantification of adsorbed and released from the matrix growth factors, and qRT-PCR for quantification of gene expression in cells grown on the matrix. Our results demonstrated that primary human oral fibroblasts, periodontal ligament, and endothelial cells exhibited increased migration toward vCM compared to control cells that migrated in the absence of the matrix. Cellular adhesive properties on vCM were significantly increased compared to controls. Growth factors TGF-ß1, PDGF-BB, FGF-2, and GDF-5 were adsorbed on vCM with great efficiency and continuously delivered in the medium after an initial burst release within hours. We observed statistically significant upregulation of genes encoding the antifibrinolytic thrombomodulin, plasminogen activator inhibitor type 1, thrombospondin 1, and thromboplastin, as well as strong downregulation of genes encoding the profibrinolytic tissue plasminogen activator, urokinase-type plasminogen activator, its receptor, and the matrix metalloproteinase 14 in cells grown on vCM. As a general trend, the stimulatory effect of the vCM on the expression of antifibrinolytic genes was synergistically enhanced by TGF-ß1, PDGF-BB, or FGF-2, whereas the strong inhibitory effect of the vCM on the expression of profibrinolytic genes was reversed by PDGF-BB, FGF-2, or GDF-5. Taken together, our data strongly support the effect of the novel vCM on fibrin clot stabilization and coagulation/fibrinolysis equilibrium, thus facilitating progression to the next stages of the soft tissue healing process.

Role of hyaluronan in regulating self-renewal and osteogenic differentiation of mesenchymal stromal cells and pre-osteoblasts.

OBJECTIVES: The aim of the study was to investigate the impact of two hyaluronan (HA) formulations on the osteogenic potential of osteoblast precursors. MATERIALS AND METHODS: Proliferation rates of HA-treated mesenchymal stromal ST2 and pre-osteoblastic MC3T3-E1 cells were determined by 5-bromo-20-deoxyuridine (BrdU) assay. Expression of genes encoding osteogenic differentiation markers, critical growth, and stemness factors as well as activation of downstream signaling pathways in the HA-treated cells were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunoblot techniques. RESULTS: The investigated HAs strongly stimulated the growth of the osteoprogenitor lines and enhanced the expression of genes encoding bone matrix proteins. However, expression of late osteogenic differentiation markers was significantly inhibited, accompanied by decreased bone morphogenetic protein (BMP) signaling. The expression of genes encoding transforming growth factor-ß1 (TGF-ß1) and fibroblast growth factor-1 (FGF-1) as well as the phosphorylation of the downstream signaling molecules Smad2 and Erk1/2 were enhanced upon HA treatment. We observed significant upregulation of the transcription factor Sox2 and its direct transcription targets and critical stemness genes, Yap1 and Bmi1, in HA-treated cells. Moreover, prominent targets of the canonical Wnt signaling pathway showed reduced expression, whereas inhibitors of the pathway were considerably upregulated. We detected decrease of active ß-catenin levels in HA-treated cells due to ß-catenin being phosphorylated and, thus, targeted for degradation. CONCLUSIONS: HA strongly induces the growth of osteoprogenitors and maintains their stemness, thus potentially regulating the balance between self-renewal and differentiation during bone regeneration following reconstructive oral surgeries. CLINICAL RELEVANCE: Addition of HA to deficient bone or bony defects during implant or reconstructive periodontal surgeries may be a viable approach for expanding adult stem cells without losing their replicative and differentiation capabilities.

Activity of two hyaluronan preparations on primary human oral fibroblasts.

BACKGROUND AND OBJECTIVE: The potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration. MATERIAL AND METHODS: Metabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing-related gene expression and activation of signaling kinases were analyzed by qRT-PCR and immunoblotting, respectively. RESULTS: The investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor-ß3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro-proliferative, pro-migratory, and pro-inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro-inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts. CONCLUSION: Both investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.

SAP domain-dependent Mkl1 signaling stimulates proliferation and cell migration by induction of a distinct gene set indicative of poor prognosis in breast cancer patients.

BACKGROUND: The main cause of death of breast cancer patients is not the primary tumor itself but the metastatic disease. Identifying breast cancer-specific signatures for metastasis and learning more about the nature of the genes involved in the metastatic process would 1) improve our understanding of the mechanisms of cancer progression and 2) reveal new therapeutic targets. Previous studies showed that the transcriptional regulator megakaryoblastic leukemia-1 (Mkl1) induces tenascin-C expression in normal and transformed mammary epithelial cells. Tenascin-C is known to be expressed in metastatic niches, is highly induced in cancer stroma and promotes breast cancer metastasis to the lung. METHODS: Using HC11 mammary epithelial cells overexpressing different Mkl1 constructs, we devised a subtractive transcript profiling screen to identify the mechanism by which Mkl1 induces a gene set co-regulated with tenascin-C. We performed computational analysis of the Mkl1 target genes and used cell biological experiments to confirm the effect of these gene products on cell behavior. To analyze whether this gene set is prognostic of accelerated cancer progression in human patients, we used the bioinformatics tool GOBO that allowed us to investigate a large breast tumor data set linked to patient data. RESULTS: We discovered a breast cancer-specific set of genes including tenascin-C, which is regulated by Mkl1 in a SAP domain-dependent, serum response factor-independent manner and is strongly implicated in cell proliferation, cell motility and cancer. Downregulation of this set of transcripts by overexpression of Mkl1 lacking the SAP domain inhibited cell growth and cell migration. Many of these genes are direct Mkl1 targets since their promoter-reporter constructs were induced by Mkl1 in a SAP domain-dependent manner. Transcripts, most strongly reduced in the absence of the SAP domain were mechanoresponsive. Finally, expression of this gene set is associated with high-proliferative poor-outcome classes in human breast cancer and a strongly reduced survival rate for patients independent of tumor grade. CONCLUSIONS: This study highlights a crucial role for the transcriptional regulator Mkl1 and its SAP domain during breast cancer progression. We identified a novel gene set that correlates with bad prognosis and thus may help in deciding the rigor of therapy.

Innate-like control of human iNKT cell autoreactivity via the hypervariable CDR3beta loop.

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3beta, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3beta loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3beta in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist alpha-linked glycolipid antigen OCH and structurally different endogenous beta-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3beta sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3beta for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3beta dependent functional hierarchy of human iNKT cells.

Differential molecular profiles and associated functionalities characterize connective tissue grafts obtained at different locations and depths in the human palate.

The present study aimed to assess the molecular profiles of subepithelial connective tissue grafts (CTGs) obtained at different locations and depths in the human palate. Sixty-four CTGs belonging to anterior deep (AD), anterior superficial (AS), posterior deep (PD), and posterior superficial (PS) groups were subjected to RNA-Sequencing and their transcriptomes were analyzed computationally. Functional correlations characterizing the CTG groups were validated by cell biological experiments using primary human palatal fibroblasts (HPFs) extracted from the CTGs. A clearly more pronounced location-dependent than depth-dependent difference between the grafts, with a minimal number of genes (4) showing no dependence on the location, was revealed. Epithelial, endothelial, and monocytic cell migration was strongly (P < 0.001) potentiated by AD- and PS-HPFs. Moreover, significantly increased expression of genes encoding C-C and C-X-C motif chemokine ligands as well as significantly (P < 0.01) activated p38 signaling suggested immunomodulatory phenotype for AD- and PS-HPFs. Increased growth factor gene expression and significantly activated (P < 0.001) Erk and Akt signaling in HPFs originating from A-CTGs implied their involvement in cell survival, proliferation, and motility. Prominent collagen-rich expression profile contributing to high mechanical stability, increased osteogenesis-related gene expression, and strongly activated (P < 0.001) Smad1/5/8 signaling characterized HPFs originating from P-CTGs. The present data indicate that in humans, differences between palatal CTGs harvested from different locations and depths appear to be location- rather than depth-dependent. Our findings provide the basis for future personalization of the therapeutic strategy by selecting an optimal graft type depending on the clinical indications.

The transcriptional regulator megakaryoblastic leukemia-1 mediates serum response factor-independent activation of tenascin-C transcription by mechanical stress.

The extracellular matrix protein tenascin-C (TNC) is up-regulated in processes influenced by mechanical stress, such as inflammation, tissue remodeling, wound healing, and tumorigenesis. Cyclic strain-induced TNC expression depends on RhoA-actin signaling, the pathway that regulates transcriptional activity of serum response factor (SRF) by its coactivator megakaryoblastic leukemia-1 (MKL1). Therefore, we tested whether MKL1 controls TNC transcription. We demonstrate that overexpression of MKL1 strongly induces TNC expression in mouse NIH3T3 fibroblasts and normal HC11 and transformed 4T1 mammary epithelial cells. Part of the induction was dependant on SRF and a newly identified atypical CArG box in the TNC promoter. Another part was independent of SRF but required the SAP domain of MKL1. An MKL1 mutant incapable of binding to SRF still strongly induced TNC, while induction of the SRF target c-fos was abolished. Cyclic strain failed to induce TNC in MKL1-deficient but not in SRF-deficient fibroblasts, and strain-induced TNC expression strongly depended on the SAP domain of MKL1. Promoter-reporter and chromatin immunoprecipitation experiments unraveled a SAP-dependent, SRF-independent interaction of MKL1 with the proximal promoter region of TNC, attributing for the first time a functional role to the SAP domain of MKL1 in regulating gene expression.

Positive Effects of Three-Dimensional Collagen-Based Matrices on the Behavior of Osteoprogenitors.

Recent research has demonstrated that reinforced three-dimensional (3D) collagen matrices can provide a stable scaffold for restoring the lost volume of a deficient alveolar bone. In the present study, we aimed to comparatively investigate the migratory, adhesive, proliferative, and differentiation potential of mesenchymal stromal ST2 and pre-osteoblastic MC3T3-E1 cells in response to four 3D collagen-based matrices. Dried acellular dermal matrix (DADM), hydrated acellular dermal matrix (HADM), non-crosslinked collagen matrix (NCM), and crosslinked collagen matrix (CCM) did all enhance the motility of the osteoprogenitor cells. Compared to DADM and NCM, HADM and CCM triggered stronger migratory response. While cells grown on DADM and NCM demonstrated proliferative rates comparable to control cells grown in the absence of a biomaterial, cells grown on HADM and CCM proliferated significantly faster. The pro-proliferative effects of the two matrices were supported by upregulated expression of genes regulating cell division. Increased expression of genes encoding the adhesive molecules fibronectin, vinculin, CD44 antigen, and the intracellular adhesive molecule-1 was detected in cells grown on each of the scaffolds, suggesting excellent adhesive properties of the investigated biomaterials. In contrast to genes encoding the bone matrix proteins collagen type I (Col1a1) and osteopontin (Spp1) induced by all matrices, the expression of the osteogenic differentiation markers Runx2, Alpl, Dlx5, Ibsp, Bglap2, and Phex was significantly increased in cells grown on HADM and CCM only. Short/clinically relevant pre-coating of the 3D biomaterials with enamel matrix derivative (EMD) or recombinant bone morphogenetic protein-2 (rBMP-2) significantly boosted the osteogenic differentiation of both osteoprogenitor lines on all matrices, including DADM and NCM, indicating that EMD and BMP-2 retained their biological activity after being released from the matrices. Whereas EMD triggered the expression of all osteogenesis-related genes, rBMP-2 upregulated early, intermediate, and late osteogenic differentiation markers except for Col1a1 and Spp1. Altogether, our results support favorable influence of HADM and CCM on the recruitment, growth, and osteogenic differentiation of the osteoprogenitor cell types. Furthermore, our data strongly support the biofunctionalization of the collagen-based matrices with EMD or rBMP-2 as a potential treatment modality for bone defects in the clinical practice.

Enhanced Wound Healing Potential of Primary Human Oral Fibroblasts and Periodontal Ligament Cells Cultured on Four Different Porcine-Derived Collagen Matrices.

Xenogenic collagen-based matrices represent an alternative to subepithelial palatal connective tissue autografts in periodontal and peri-implant soft tissue reconstructions. In the present study, we aimed to investigate the migratory, adhesive, proliferative, and wound-healing potential of primary human oral fibroblasts (hOF) and periodontal ligament cells (hPDL) in response to four commercially available collagen matrices. Non-crosslinked collagen matrix (NCM), crosslinked collagen matrix (CCM), dried acellular dermal matrix (DADM), and hydrated acellular dermal matrix (HADM) were all able to significantly enhance the ability of hPDL and hOF cells to directionally migrate toward the matrices as well as to efficiently repopulate an artificially generated wound gap covered by the matrices. Compared to NCM and DADM, CCM and HADM triggered stronger migratory response. Cells grown on CCM and HADM demonstrated significantly higher proliferative rates compared to cells grown on cell culture plastic, NCM, or DADM. The pro-proliferative effect of the matrices was supported by expression analysis of proliferative markers regulating cell cycle progression. Upregulated expression of genes encoding the adhesive molecules fibronectin, vinculin, CD44 antigen, and the intracellular adhesive molecule-1 was detected in hPDL and hOF cells cultured on each of the four matrices. This may be considered as a prerequisite for good adhesive properties of the four scaffolds ensuring proper cell-matrix and cell-cell interactions. Upregulated expression of genes encoding TGF-ß1 and EGF growth factors as well as MMPs in cells grown on each of the four matrices provided support for their pro-proliferative and pro-migratory abilities. The expression of genes encoding the angiogenic factors FGF-2 and VEGF-A was dramatically increased in cells grown on DADM and HADM only, suggesting a good basis for accelerated vascularization of the latter. Altogether, our results support favorable influence of the investigated collagen matrices on the recruitment, attachment, and growth of cell types implicated in oral soft tissue regeneration. Among the four matrices, HADM has consistently exhibited stronger positive effects on the oral cellular behavior. Our data provide solid basis for future investigations on the clinical application of the collagen-based matrices in surgical periodontal therapy.

Adsorption and Release of Growth Factors from Four Different Porcine-Derived Collagen Matrices.

Xenogeneic acellular collagen matrices represent a safe alternative to autologous soft tissue transplants in periodontology and implant dentistry. Here, we aimed to investigate the adsorption and release of growth factors from four porcine-derived collagen matrices using enzyme-linked immunosorbent assay. Non-crosslinked collagen matrix (NCM), crosslinked collagen matrix (CCM), dried acellular dermal matrix (DADM), and hydrated acellular dermal matrix (HADM) adsorbed each of the following growth factors, TGF-ß1, FGF-2, PDGF-BB, GDF-5 and BMP-2, with an efficiency close to 100%. Growth factor release for a 13-day period was in the range of 10-50% of the adsorbed protein, except for the BMP-2 release that was in the range of 5-7%. Generally, protein release occurred in two phases. Phase I was arbitrary defined by the highest release from the matrices, usually within 24 h. Phase II, spanning the period immediately after the peak release until day 13, corresponded to the delayed release of the growth factors from the deeper layers of the matrices. HADM showed significantly (P < 0.001) higher TGF-ß1, FGF-2, and PDGF-BB release in phase II, compared to the rest of the matrices. NCM exhibited significantly (P < 0.001) higher FGF-2 release in phase II, compared to CCM and DADM as well as a characteristic second peak in PDGF-BB release towards the middle of the tested period. In contrast to NCM and HADM, CCM and DADM showed a gradual and significantly higher release of GDF-5 in the second phase. Several burst releases of BMP-2 were characteristic for all matrices. The efficient adsorption and sustained protein release in the first 13 days, and the kinetics seen for HADM, with a burst release within hours and high amount of released growth factor within a secondary phase, may be beneficial for the long-term tissue regeneration following reconstructive periodontal surgery.

Development and characterization of a triple combination gene therapy vector inhibiting HIV-1 multiplication.

BACKGROUND: RNA-based approaches are promising for long-term gene therapy against HIV-1. They can target virtually any step of the viral replication cycle. It is also possible to combine anti-HIV-1 transgenes targeting different facets of HIV replication to compensate for limitations of any individual construct, maximizing efficacy and decreasing chances of escape mutations. We have previously developed two strategies to inhibit HIV-1 multiplication. One was a short hairpin RNA targeting the host factor cyclophilin A implicated in HIV-1 replication. Additionally, an antisense derivative of U7 small nuclear RNA was designed to induce the skipping of the HIV-1 Tat and Rev internal exons. RESULTS: In the present study, we have established an additional tRNAval promoter-driven shRNA against the coding sequence of viral infectivity factor. When human T-cell lines or primary CD4+ T cells are transduced with a triple lentiviral vector encoding these three therapeutic RNAs, HIV-1 multiplication is very efficiently suppressed. Moreover, all three therapeutic RNAs exhibit antiviral effects at early stages of the viral replication cycle (i.e. prior to viral cDNA integration or gene expression). CONCLUSIONS: These findings make this triple lentiviral vector an attractive candidate for a gene therapy against HIV/AIDS.

Bone-conditioned medium contributes to initiation and progression of osteogenesis by exhibiting synergistic TGF-ß1/BMP-2 activity.

Guided bone regeneration (GBR) often utilizes a combination of autologous bone grafts, deproteinized bovine bone mineral (DBBM), and collagen membranes. DBBM and collagen membranes pre-coated with bone-conditioned medium (BCM) extracted from locally harvested autologous bone chips have shown great regenerative potential in GBR. However, the underlying molecular mechanism remains largely unknown. Here, we investigated the composition of BCM and its activity on the osteogenic potential of mesenchymal stromal cells. We detected a fast and significant (P < 0.001) release of transforming growth factor-ß1 (TGF-ß1) from autologous bone within 10 min versus a delayed bone morphogenetic protein-2 (BMP-2) release from 40 min onwards. BCMs harvested within short time periods (10, 20, or 40 min), corresponding to the time of a typical surgical procedure, significantly increased the proliferative activity and collagen matrix production of BCM-treated cells. Long-term (1, 3, or 6 days)-extracted BCMs promoted the later stages of osteoblast differentiation and maturation. Short-term-extracted BCMs, in which TGF-ß1 but no BMP-2 was detected, reduced the expression of the late differentiation marker osteocalcin. However, when both growth factors were present simultaneously in the BCM, no inhibitory effects on osteoblast differentiation were observed, suggesting a synergistic TGF-ß1/BMP-2 activity. Consequently, in cells that were co-stimulated with recombinant TGF-ß1 and BMP-2, we showed a significant stimulatory and dose-dependent effect of TGF-ß1 on BMP-2-induced osteoblast differentiation due to prolonged BMP signaling and reduced expression of the BMP-2 antagonist noggin. Altogether, our data provide new insights into the molecular mechanisms underlying the favorable outcome from GBR procedures using BCM, derived from autologous bone grafts.

Mechanism of irradiation-induced mammary cancer metastasis: A role for SAP-dependent Mkl1 signaling.

Radiotherapy is a standard treatment after conservative breast cancer surgery. However, cancers relapsing within a previously irradiated area have an increased probability to metastasize. The mechanisms responsible for this aggressiveness remain unclear. Here, we used the clinically relevant 4T1 breast cancer model mimicking aggressive local relapse after radiotherapy to identify differences between tumors grown in untreated versus preirradiated mammary glands. Tumors grown within preirradiated beds were highly enriched in transcripts encoding collagens and other proteins building or modifying the extracellular matrix, such as laminin-332, tenascins, lysyl oxidases and matrix metalloproteinases. Type I collagen, known to directly contribute to tissue stiffening, and the pro-metastatic megakaryoblastic leukemia-1 (Mkl1) target gene tenascin-C were further investigated. Mammary tissue preirradiation induced Mkl1 nuclear translocation in the tumor cells in vivo, indicating activation of Mkl1 signaling. Transcript profiling of cultured 4T1 cells revealed that the majority of the Mkl1 target genes, including tenascin-C, required serum response factor (SRF) for their expression. However, application of dynamic strain or matrix stiffness to 4T1 cells converted the predominant SRF/Mkl1 action into SAP domain-dependent Mkl1 signaling independent of SRF, accompanied by a switch to SAP-dependent tumor cell migration. 4T1 tumors overexpressing intact Mkl1 became more metastatic within preirradiated beds, while tumors expressing Mkl1 lacking the SAP domain exhibited impaired growth and metastatic spread, and decreased Mkl1 target gene expression. Thus, we identified SAP-dependent Mkl1 signaling as a previously unrecognized mediator of aggressive progression of mammary tumors locally relapsing after radiotherapy, and provide a novel signaling pathway for therapeutic intervention.

Megakaryoblastic leukemia protein-1 (MKL1): Increasing evidence for an involvement in cancer progression and metastasis.

Megakaryoblastic leukemia protein-1 (MKL1), also termed MAL, MRTF-A, and BSAC, belongs to the MRTF family of transcription factors that share evolutionary conserved domains required for actin-binding, homo- and heterodimerization, high-order chromatin organization and transcriptional activation. MKL1 regulates many processes, including muscle cell differentiation, cardiovascular development, remodeling of neuronal networks in the developing and adult brain, megakaryocytic differentiation and migration, modulation of cellular motile functions and epithelial-mesenchymal transition. Moreover, deregulation by genetic alterations and/or altered expression of MKL1 can contribute to a number of pathological processes such as coronary artery disease, sarcopenia, acute megakaryoblastic leukemia, and cancer. In this article, we review the structure, regulation and biological functions of MKL1. In addition, we discuss recent evidence that strongly suggests a dual role for MKL1 in oncogenic mechanisms, as a tumor-promoting or tumor-suppressing molecule. Future studies will be necessary to evaluate the potential clinical implications of MKL1 expression and activation in cancer.

Inhibition of HIV-1 multiplication by a modified U7 snRNA inducing Tat and Rev exon skipping.

The HIV-1 regulatory proteins Tat and Rev are encoded by multiply spliced mRNAs that differ by the use of alternative 3' splice sites at the beginning of the internal exon. If these internal exons are skipped, the expression of these genes, and hence HIV-1 multiplication, should be inhibited. We have previously developed a strategy, based on antisense derivatives of U7 small nuclear RNA, that allows us to induce the skipping of an internal exon in virtually any gene. Here, we have successfully applied this approach to induce a partial skipping of the Tat, Rev (and Nef) internal exons. Three functional U7 constructs were subcloned into a lentiviral vector. Two of them strongly reduced the efficiency of lentiviral particle production compared to vectors carrying either no U7 insert or unrelated U7 cassettes. This defect could be partly or fully compensated by coexpressing Rev from an unspliced mRNA in the producing cell line. Upon stable transduction into CEM-SS or CEM T-lymphocytes, the most efficient of these constructs inhibits HIV-1 multiplication. Although the inhibition is not complete, it is more efficient in combination with another mechanism inhibiting HIV multiplication. Therefore, this new approach targeting HIV-1 regulatory genes at the level of pre-mRNA splicing, in combination with other antiviral strategies, may be a useful new tool in the fight against HIV/AIDS.