Multiscale analysis of Klf10's impact on the passive mechanical properties of murine skeletal muscle.

Skeletal muscle is a hierarchical structure composed of multiple organizational scales. A major challenge in the biomechanical evaluation of muscle relates to the difficulty in evaluating the experimental mechanical properties at the different organizational levels of the same tissue. Indeed, the ability to integrate mechanical properties evaluated at various levels will allow for improved assessment of the entire tissue, leading to a better understanding of how changes at each level evolve over time and/or impact tissue function, especially in the case of muscle diseases. Therefore, the purpose of this study was to analyze a genetically engineered mouse model (Klf10 KO: Krüppel-Like Factor 10 knockout) with known skeletal muscle defects to compare the mechanical properties with wild-type (WT) controls at the three main muscle scales: the macroscopic (whole muscle), microscopic (fiber) and submicron (myofibril) levels. Passive mechanical tests (ramp, relaxation) were performed on two types of skeletal muscle (soleus and extensor digitorum longus (EDL)). Results of the present study revealed muscle-type specific behaviors in both genotypes only at the microscopic scale. Interestingly, loss of Klf10 expression resulted in increased passive properties in the soleus but decreased passive properties in the EDL compared to WT controls. At the submicron scale, no changes were observed between WT and Klf10 KO myofibrils for either muscle; these results demonstrate that the passive property differences observed at the microscopic scale (fiber) are not caused by sarcomere intrinsic alterations but instead must originate outside the sarcomeres, likely in the collagen-based extracellular matrix. The macroscopic scale revealed similar passive mechanical properties between WT and Klf10 KO hindlimb muscles. The present study has allowed for a better understanding of the role of Klf10 on the passive mechanical properties of skeletal muscle and has provided reference data to the literature which could be used by the community for muscle multiscale modeling.

The Effect of Freezing Time on Muscle Fiber Mechanical Properties.

The purpose of this study was to investigate the effect of freezing time on the functional behavior of mouse muscle fibers. Passive mechanical tests were performed on single soleus muscle fibers from fresh (0 month) and preserved (stored at -20°C for 3, 6, 9 and 12 months) 3 month old mice. The Young's modulus and the dynamic and the static stresses were measured. A viscoelastic Hill model of 3rd order was used to fit the experimental relaxation test data. The statistical analysis corresponding to the elastic modulus of single muscle fibers did not differ when comparing fresh and stored samples for 3 and 6 months at -20 °C. From 9 months, fibers were less resistant and the mechanical properties were damaged. The primary goal of this study was to complete the gold standard process of muscle fiber preservation for subsequent mechanical property studies. We have demonstrated that muscle fibers can be stored at -20°C for up to 6 months without altering their mechanical properties.

KLF10 loss in the pancreas provokes activation of SDF-1 and induces distant metastases of pancreatic ductal adenocarcinoma in the KrasG12D p53flox/flox model.

Krüppel-like transcription factor 10 (KLF10), also named as TIEG1, plays essential roles in mediating transforming growth factor beta (TGFß) signaling and has been shown to function as a tumor suppressor in multiple cancer types. However, its roles in mediating cancer progression in vivo have yet to be fully characterized. Here, we have employed two well-characterized Pdx-1CreLSL-KrasG12D and Pdx-1CreLSL-KrasG12Dp53L/L pancreatic cancer models to ablate KLF10 expression and determine the impact of KLF10 deletion on tumor development and progression. We show that loss of KLF10 cooperates with KrasG12D leading to an invasive and widely metastatic phenotype of pancreatic ductal adenocarcinoma (PDAC). Mechanistically, loss of KLF10 in PDAC is shown to increase distant metastases and cancer stemness through activation of SDF-1/CXCR4 and AP-1 pathways. Furthermore, we demonstrate that targeting the SDF-1/CXCR4 pathway in the context of KLF10 deletion substantially suppresses PDAC progression suggesting that inhibition of this pathway represents a novel therapeutic strategy for PDAC treatment.

TGFbeta inducible early gene-1 directly binds to, and represses, the OPG promoter in osteoblasts.

TGFbeta inducible early gene-1 (TIEG) is a member of the Krüppel-like family of transcription factors (KLF10) that plays an important role in TGFbeta mediated Smad signaling. In order to better understand the role of TIEG in bone, we generated TIEG knockout (KO) mice. Calvarial osteoblasts (OBs) isolated from these mice exhibit a reduced ability to support osteoclastogenesis in vitro. Gene expression studies revealed decreased receptor activator of NF-kappaB ligand (RANKL) and increased osteoprotegerin (OPG) expression in TIEG KO OBs, suggesting a potential role for TIEG in regulating the expression of these genes. Since OPG and RANKL are two important regulators of osteoclast (OC) differentiation, we sought to determine if TIEG directly regulates their expression. Luciferase constructs, containing fragments of either the mouse OPG promoter (-1486 to +133 bp) or the RANKL promoter (-2000 to +1 bp) were each cloned into the pGL3 basic reporter vector and transiently transfected into TIEG KO calvarial OBs with and without a TIEG expression vector. No significant changes in the activity of the RANKL promoter were detected in the presence of TIEG. However, OPG promoter activity was inhibited in the presence of TIEG protein suggesting that TIEG directly represses the expression of OPG in OBs. In order to determine the region of this promoter through which TIEG acts, sequential 5'-deletion constructs were generated. Transient transfection of these constructs revealed that the TIEG regulatory element(s) reside within a 200 bp region of the OPG promoter. Transient ChIP analyses, using a TIEG-specific antibody, revealed that TIEG binds to this region of the OPG promoter. Since we have previously shown that TIEG regulates target gene expression through Sp-1 sites, we examined this region of the OPG promoter for potential TIEG binding elements and identified four potential Sp-1 binding sites. Site-directed mutagenesis was used to determine if TIEG utilizes these Sp-1 elements to regulate the activity of the OPG promoter. The data demonstrate that two Sp-1 sites are likely to be involved in TIEG's repression of the OPG promoter. Taken together, these results confirm that TIEG directly binds to and inhibits OPG promoter activity in OBs, partially explaining the inability of TIEG KO OBs to fully support osteoclast differentiation.

TIEG-null mice display an osteopenic gender-specific phenotype.

TGFbeta inducible early gene-1 (TIEG) was originally cloned from human osteoblasts (OB) and has been shown to play an important role in TGFbeta/Smad signaling, regulation of gene expression and OB growth and differentiation. To better understand the biological role of TIEG in the skeleton, we have generated congenic TIEG-null (TIEG(-/-)) mice in a pure C57BL/6 background. Through the use of DXA and pQCT analysis, we have demonstrated that the femurs and tibias of two-month-old female TIEG(-/-) mice display significant decreases in total bone mineral content, density, and area relative to wild-type (WT) littermates. However, no differences were observed for any of these bone parameters in male mice. Further characterization of the bone phenotype of female TIEG(-/-) mice involved mechanical 3-point bending tests, micro-CT, and histomorphometric analyses of bone. The 3-point bending tests revealed that the femurs of female TIEG(-/-) mice have reduced strength with increased flexibility compared to WT littermates. Micro-CT analysis of femurs of two-month-old female TIEG(-/-) mice revealed significant decreases in cortical bone parameters compared to WT littermates. Histomorphometric evaluation of the distal femur revealed that female TIEG(-/-) mice also display a 31% decrease in cancellous bone area, which is primarily due to a decrease in trabecular number. At the cellular level, female TIEG(-/-) mice exhibit a 42% reduction in bone formation rate which is almost entirely due to a reduction in double labeled perimeter. Differences in mineral apposition rate were not detected between WT and TIEG(-/-) mice. Taken together, these findings suggest that female TIEG(-/-) mice are osteopenic mainly due to a decrease in the total number of functional/mature OBs.

Estrogen-TGFbeta cross-talk in bone and other cell types: role of TIEG, Runx2, and other transcription factors.

It is well established that E(2) and TGFbeta have major biological effects in multiple tissues, including bone. The signaling pathways through which these two factors elicit their effects are well documented. However, the interaction between these two pathways and the potential consequences of cross-talk between E(2) and TGFbeta continue to be elucidated. In this prospectus, we present known and potential roles of TIEG, Runx2, and other transcription factors as important mediators of signaling between these two pathways.

Identification and functional clustering of global gene expression differences between age-related cataract and clear human lenses and aged human lenses.

We have examined the gene expression profiles of young, old and cataractous human lenses in order to differentiate those gene expression changes specific for cataract from those also associated with lens aging. Differentially expressed transcripts were identified by oligonucleotide microarray analysis and clustered according to their known functions. Four hundred and twelve transcripts that are increased and 919 transcripts that are decreased were identified at the 2-fold or greater level between epithelia isolated from cataract relative to clear lenses while 182 transcripts that are increased and 547 transcripts that are decreased were identified at the 2-fold or greater level between young and old lens epithelia. Comparison of the cataract gene expression changes with those detected in lens aging revealed that only 3 transcripts exhibited similar trends in gene expression. These data suggest that cataract- and age-specific changes in gene expression do not overlap and provide evidence for multiple cataract- and age-specific gene expression changes in the human lens.