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Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells.

ABSTRACT: Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgehog (Shh-N) significantly increased in vitro osteogenic differentiation in mouse ASCs, by all markers examined (*p < 0.01). Concomitantly, Shh-N abrogated adipogenic differentiation, by all markers examined (*p < 0.01). Conversely, blockade of endogenous Hedgehog signaling, with the Hedgehog antagonist cyclopamine, enhanced adipogenesis at the expense of osteogenesis. We next translated these results to a mouse model of appendicular skeletal regeneration. Using quantitative real-time polymerase chain reaction and in situ hybridization, we found that skeletal injury (a monocortical 1 mm defect in the tibia) results in a localized increase in Hedgehog signaling. Moreover, grafting of ASCs treated with Shh-N resulted in significantly increased bone regeneration within the defect site. In conclusion, Hedgehog signaling enhances the osteogenic differentiation of mouse ASCs, at the expense of adipogenesis. These data suggest that Hedgehog signaling directs the lineage differentiation of mesodermal stem cells and represents a promising strategy for skeletal tissue regeneration.

SUBMITTER: James AW

PROVIDER: S-EPMC2947454 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells.

James Aaron W AW Leucht Philipp P Levi Benjamin B Carre Antoine L AL Xu Yue Y Helms Jill A JA Longaker Michael T MT

Tissue engineering. Part A 20100801 8

Adipose-derived stromal cells (ASCs) present a great potential for tissue engineering, as they are capable of differentiating into osteogenic and adipogenic cell types, among others. In this study, we examined the role of Hedgehog signaling in the balance of osteogenic and adipogenic differentiation in mouse ASCs. Results showed that Hedgehog signaling increased during early osteogenic differentiation (Shh, Ptc1, and Gli1), but decreased during adipogenic differentiation. N-terminal Sonic Hedgeh ...[more]

PMID: 20367246

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Project description:Previous studies have demonstrated that myocardin-related transcription factor A (MRTF-A) generates a link between the dynamics of the actin cytoskeleton and gene expression with its coregulator, serum response factor (SRF). MRTF-A has also been suggested as a regulator of stem cell differentiation. However, the role of MRTF-A in human mesenchymal stem cell differentiation remains understudied. We aimed to elucidate whether MRTF-A is a potential regulator of human adipose stem cell (hASC) differentiation towards adipogenic and osteogenic lineages. To study the role of MRTF-A activity in the differentiation process, hASCs were cultured in adipogenic and osteogenic media supplemented with inhibitor molecules CCG-1423 or CCG-100602 that have been shown to block the expression of MRTF-A/SRF-activated genes. Our results of image-based quantification of Oil Red O stained lipid droplets and perilipin 1 staining denote that MRTF-A inhibition enhanced the adipogenic differentiation. On the contrary, MRTF-A inhibition led to diminished activity of an early osteogenic marker alkaline phosphatase, and export of extracellular matrix (ECM) proteins collagen type I and osteopontin. Also, quantitative Alizarin Red staining representing ECM mineralization was significantly decreased under MRTF-A inhibition. Image-based analysis of Phalloidin staining revealed that MRTF-A inhibition reduced the F-actin formation and parallel orientation of the actin filaments. Additionally, MRTF-A inhibition affected the protein amounts of α-smooth muscle actin (α-SMA), myosin light chain (MLC), and phosphorylated MLC suggesting that MRTF-A would regulate differentiation through SRF activity. Our results strongly indicate that MRTF-A is an important regulator of the balance between osteogenesis and adipogenesis of hASCs through its role in mediating the cytoskeletal dynamics. These results provide MRTF-A as a new interesting target for guiding the stem cell differentiation in tissue engineering applications for regenerative medicine.

Project description:Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. It is typically detected at an advanced stage, at which the therapeutic options are very limited. One remarkable feature of PDAC that contributes to its resilience to treatment is the extreme stromal activation seen in these tumors. Often, the vast majority of tumor bulk consists of non-tumor cells that together provide a tumor-promoting environment. One of the signals that maintains and activates the stroma is the developmental protein Sonic Hedgehog (SHH). As the disease progresses, tumor cells produce increasing amounts of SHH, which activates the surrounding stroma to aid in tumor progression. To better understand this response and identify targets for inhibition, we aimed to elucidate the proteins that mediate the SHH-driven stromal response in PDAC. For this a novel mixed-species coculture model was set up in which the cancer cells are human, and the stroma is modeled by mouse fibroblasts. In conjunction with next-generation sequencing we were able to use the sequence difference between these species to genetically distinguish between the epithelial and stromal responses to SHH. The stromal SHH-dependent genes from this analysis were validated and their relevance for human disease was subsequently determined in two independent patient cohorts. In non-microdissected tissue from PDAC patients, in which a large amount of stroma is present, the targets were confirmed to associate with tumor stroma versus normal pancreatic tissue. Patient survival analysis and immunohistochemistry identified CDA, EDIL3, ITGB4, PLAUR and SPOCK1 as SHH-dependent stromal factors that are associated with poor prognosis in PDAC patients. Summarizing, the presented data provide insight into the role of the activated stroma in PDAC, and how SHH acts to mediate this response. In addition, the study has yielded several candidates that are interesting therapeutic targets for a disease for which treatment options are still inadequate.

Dataset Information

Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells.

Publications

Sonic Hedgehog influences the balance of osteogenesis and adipogenesis in mouse adipose-derived stromal cells.

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