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 twoHAformulations 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 uponHAtreatment. To uncover the mechanisms wherebyHAinfluences cellular behavior, wound healing‐related gene expression and activation of signaling kinases were analyzed byqRT‐PCRand immunoblotting, respectively.Results: The investigatedHAformulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding typeIIIcollagen and transforming growth factor‐β3, characteristic of scarless wound healing. TheHAs 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 ofHAon 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 theHAs exert their effects on oral fibroblasts.Conclusion: Both investigatedHAformulations 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, theHAs are not likely to impair the healing process by prolonging inflammation or causing excessiveMMPexpression at the repair site.

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, CDR3β, 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 CDR3β 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 CDR3β 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 α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β 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, CDR3β dependent functional hierarchy of human iNKT cells. Our immune system uses randomly modified T-cell receptors (TCRs) to adapt its discriminative capacity to rapidly changing pathogens. The T-cell receptor (TCR) has six flexible, variable peptide loops that make contact with antigens presented to them on the surface of other cells. Invariant Natural Killer T-cells (iNKT) are regulatory T-cells with a unique type of TCR (iNKT-TCR) that recognizes lipid antigens presented by specific MHC-like molecules known as CD1d. In human iNKT-TCRs, only one of the six loops, CDR3beta, is variable. By comparing how different human iNKT clones bind and react to different CD1d-lipid complexes we uncover the existence of a hierarchical order of the human iNKT cell repertoire in which strongly CD1d-binding clones are autoreactive while weak CD1d-binding clones are non-autoreactive. Direct measurements of iNKT-TCR binding to CD1d using surface plasmon resonance recapitulated this hierarchy at the protein level. The data show that variation in the CDR3beta loop conveys dramatic differences in human iNKT TCR affinity that are independent of the CD1d bound ligand. Thus the CDR3beta loop provides the structural basis for the functional hierarchy of the human iNKT repertoire. We postulate that during the life-course, CDR3beta-dependent asymmetrical activation of different human iNKT clones leads to a bias in the iNKT repertoire, and this could result in age-dependent defects of iNKT-mediated immune regulation in later life.

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.—Asparuhova, M. B., Ferralli, J., Chiquet, M., Chiquet‐Ehrismann, R. The transcriptional regulator megakaryoblastic leukemia‐1 mediates serum response factor‐independent activation of tenascin‐C transcription by mechanical stress. FASEB J. 25, 3477–3488 (2011). www.fasebj.org

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-β1 (TGF-β1) 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 β-catenin levels in HA-treated cells due to β-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.

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.