Transforming growth factor-beta-regulated miR-24 promotes skeletal muscle differentiation.

MicroRNAs (miRNAs) have recently been proposed as a versatile class of molecules involved in regulation of a variety of biological processes. However, the role of miRNAs in TGF-beta-regulated biological processes is poorly addressed. In this study, we found that miR-24 was upregulated during myoblast differentiation and could be inhibited by TGF-beta1. Using both a reporter assay and Northern blot analysis, we showed that TGF-beta1 repressed miR-24 transcription which was dependent on the presence of Smad3 and a Smads binding site in the promoter region of miR-24. TGF-beta1 was unable to inhibit miR-24 expression in Smad3-deficient myoblasts, which exhibited accelerated myogenesis. Knockdown of miR-24 led to reduced expression of myogenic differentiation markers in C2C12 cells, while ectopic expression of miR-24 enhanced differentiation, and partially rescued inhibited myogenesis by TGF-beta1. This is the first study demonstrating a critical role for miRNAs in modulating TGF-beta-dependent inhibition of myogenesis, and provides a novel mechanism of the genetic regulation of TGF-beta signaling during skeletal muscle differentiation.

Capn8 promoter directs the expression of Cre recombinase in gastric pit cells of transgenic mice.

Gastric pit cells are high-turnover epithelial cells of the gastric mucosa. They secrete mucus to protect the gastric epithelium from acid and pepsin. To investigate the genetic mechanisms underlying the physiological functions of gastric pit cells, we generated a transgenic mouse line, namely, Capn8-Cre, in which the expression of Cre recombinase was controlled by the promoter of the intracellular Ca(2+)-regulated cysteine protease calpain-8. To test the tissue distribution and excision activity of Cre recombinase, the Capn8-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple-tissue PCR and LacZ staining demonstrated that Capn8-Cre transgenic mouse expressed Cre recombinase in the gastric pit cells. Cre recombinase activity was also detected in the liver and skin tissues. These data suggest that the Capn8-Cre mouse line described here could be used to dissect gene function in gastric pit cells.

[SMAD3 inhibits the expression of MMP-2 in mouse embryonic fibroblasts].

SMAD3 is one of the receptor-activated SMADs which are important in TGF-beta signal transduction. Smad3-null mice show accelerated cutaneous wound healing compared with wild-type mice. In this work, we investigated the functions and the mechanism of Smad3-mediated TGF-beta signal on matrix metaloproteinase-2 (MMP-2) in mouse fibroblast. We found that MMP-2 at wound bed was expressed earlier in Smad3-null mice than that in wild type and heterozygotes. In the sera of wounding mice, the activity of MMP-2 was also remarkably higher in Smad3-null mice than that in the other two. The embryonic fibroblasts were separated from Smad3 knockout mice to test the function of Smad3 in modulating the expression of MMP-2. The results showed that the expression and activity of MMP-2 in Smad3-null fibroblasts were higher than those in wild type cells. TGF-beta1 could increase the MMP-2 activities in both Smad3 mutant and wild type fibroblasts. The expression and activity of MMP-2 were inhibited by over expression of SMAD3 in Smad3-null fibroblasts, while the expression and activity of MMP-2 were increased by over expression of anti-sense Smad3 in wild type cells. All these results showed that SMAD3 inhibited the expression of MMP-2 in mouse embryonic fibroblasts.

Establishment of Smad2 conditional gene targeting mice based on the Cre-LoxP system.

Smads is a new gene family in transforming growth factor-beta (TGF- beta) signaling pathway. Smad2 mutated in multiple human tumors and may be a candidate tumor suppressor gene. Targeted disruption of murine Smad2 gene resulted in embryonic lethality at E6.5. To study the function of Smad2 in vertebrate organgenesis and tumorigenesis, we constructed the Smad2 conditional targeting vector in which two LoxP sequences were placed to flank the sequences encoding the C terminal functional domain of Smad2. The validity of the LoxP sites in the targeting construct was tested in E. coli that express the Cre recombinase constitutively. The vector was electroporated into ES cells and 3 targeted ES cell clones were obtained by Southern blot screening. Targeted ES cells were introduced into C57BL/6J blastocysts by microinjection to generate germ-line chimeras. Genotyping analysis showed that 2 progeny among these chimeras carried the Smad2 conditional targeted allele. The establishment of Smad2 conditional gene targeting mouse has laid a solid foundation for producing the tissue specific Smad2 gene knockout mice.

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice.

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid. To study the function of gastric parietal cells during gastric epithelium homeostasis, we generated a transgenic mouse line, namely, Atp4b-Cre, in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse (-subunit of H(+)-, K(+)-ATPase gene (Atp4b). In order to test the tissue distribution and excision activity of Cre recombinase in vivo, the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles (Smad4(Co/Co)). Multiple-tissue PCR of Atp4b-Cre;Smad4(Co/+) mice revealed that the recombination only happened in the stomach. As indicated by LacZ staining, ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells. These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.

Smad5 haploinsufficiency leads to hair cell and hearing loss.

The Smads are a group of related intracellular proteins critical for transmitting the signals to the nucleus from the transforming growth factor-beta superfamily at the cell surface. Knockout of the Smad5 is embryonic lethal. However, the Smad5 knockout of single allele (+/-) could survive. We used Smad5 heterozygous knockout (+/-) to determine the role of Smad5 in the development of inner ear morphology and function. In situ hybridization showed that Smad5 was expressed predominantly in hair cells, spiral ganglion, and supporting cells. Measurements of hearing thresholds using auditory brainstem response showed that Smad5 defect resulted in progressive hearing loss between 4 and 24 weeks after birth. Morphological examination revealed apoptosis in the inner ear, with significant loss of outer hair cells in adult Smad5 mutant mice. Our results indicated that deficiency in the Smad5-mediated signaling resulted in apoptosis of hair cells, suggesting Smad5 is a gene that may be related with presbycusis.

Disruption of Smad5 gene induces mitochondria-dependent apoptosis in cardiomyocytes.

Our previous studies have shown that SMAD5, an important intracellular mediator of transforming growth factor beta (TGF-beta) family, is required for normal development of the cardiovascular system in vivo. In the current study, we reported that the lack of the Smad5 gene resulted in apoptosis of cardiac myocytes in vivo. To further investigate the mechanism of the Smad5 gene in cardiomyocyte apoptosis, the embryonic stem (ES) cell differentiation system was employed. We found that the myotubes that differentiated from the homozygous Smad5ex6/ex6 mutant ES cells underwent collapse and degeneration during the late stages of in vitro differentiation, mimicking the in vivo observation. By electron microscopy, abnormal swollen mitochondria were observed in cardiomyocytes both from Smad5-deficient embryos and from ES-differentiated cells. There was also a significant reduction in mitochondrial membrane potential (Deltapsi m) and a leakage of cytochrome c from mitochondria into the cytosol of myocytes differentiated from Smad5 mutant ES cells. The expression of p53 and p21 was found to be elevated in the differentiated Smad5 mutant myocytes, and this was accompanied by an up-regulation in caspase 3 expression. These results suggest that the Smad5-mediated TGF-beta signals may protect cardiomyocytes from apoptosis by maintaining the integrity of the mitochondria, probably through suppression of p53 mediated pathways.

Disruption of Smad5 gene leads to enhanced proliferation of high-proliferative potential precursors during embryonic hematopoiesis.

SMAD proteins are downstream signal transducers of the transforming growth factor beta (TGF-beta) superfamily, which serve as pleiotropic regulators in embryonic and adult hematopoiesis. SMAD5, initially considered to mediate bone morphogenetic proteins (BMPs) signals, can also transduce the inhibitory signal of TGF-beta1 on proliferation of hematopoietic progenitors derived from human bone marrow. To define its specific role in regulation of primitive multipotential progenitors during early embryonic hematopoiesis, we examined Smad5(-/-) yolk sacs at E9.0 to 9.5 and detected an elevated number of high-proliferative potential colony-forming cells (HPP-CFCs) with enhanced replating potential. To exclude the possible influence of microenvironmental deficit on embryonic hematopoiesis in vivo, we performed in vitro embryonic stem (ES) cell differentiation assay and investigated the HPP-CFCs in particular. Smad5(-/-) embryoid bodies (EBs) contained an elevated number of blast colony-forming cells (BL-CFCs), the in vitro equivalent of hemangioblast, in contrast to reduced proliferation of primitive erythroid precursors (Ery/Ps) within the mutant EBs. More importantly, profoundly increased frequency of HPP-CFCs, featured with a gene-dosage effect, was detected within day 6 Smad5(-/-) EBs compared with the wild type. In addition, Smad5(-/-) HPP-CFCs displayed enhanced self-renewal capacity and decreased sensitivity to TGF-beta1 inhibition, suggesting a critical role of Smad5 in TGF-beta1 regulation of embryonic HPP-CFCs. Consistently, reverse transcription-polymerase chain reaction analysis detected alterations of the transcription factors including GATA-2 and AML1 as well as cytokine receptors in Smad5(-/-) HPP-CFC colonies. Together, these data define an important function of SMAD5 in negative regulation of high-proliferative potential precursors during embryonic hematopoiesis.