Biomedical subjects

Tamara C Otto

Publications and source records attributed to Tamara C Otto.

8 recordsLinked to original sources

PubMed2006

BMP-4 treatment of C3H10T1/2 stem cells blocks expression of MMP-3 and MMP-13.

Microarray gene expression profiling was used to identify bone morphogenetic protein-4 (BMP-4) responsive factors involved in late stages of adipocyte commitment in C3H10T1/2 cells. The analysis revealed that the matrix metalloproteinase-3 (MMP-3) gene decreased 100-fold after BMP-4 treatment, and expression of MMP-13 decreased 19.5-fold. Uncommitted C3H10T1/2 cells exhibit dramatic up-regulation of MMP-3 and MMP-13 genes as cells become confluent. Real-time RT-PCR demonstrated that BMP-4 blocks expression of both transcripts. Likewise, a stable committed preadipocyte line derived from C3H10T1/2 cells did not express MMP-3 or MMP-13 at confluence, despite never receiving BMP-4. Active forms of both proteins were detected in media from confluent C3H10T1/2 cells but not in BMP-4 treated cells. Addition of BMP-4 to confluent C3H10T1/2 cells repressed the expression of both genes but did not induce adipocyte differentiation. The findings indicate that BMP-4-induced down-regulation of MMP-3 and MMP-13 is associated with commitment, but is insufficient to induce adipogenesis.

3T3-L1 Cells↗

PubMed2006

Stable stem cell commitment to the adipocyte lineage by inhibition of DNA methylation: role of the BMP-4 gene.

Previous studies showed that exposure of C3H10T1/2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1/2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. Detailed studies performed with a cloned committed subline, the A33 line, verified stable adipocyte lineage determination in the absence of exogenous BMP-4. Remarkably, this cell line expresses and secretes BMP-4 during proliferation in the same time window that exogenous BMP-4 must be added to naïve 10T1/2 cells to induce maximal adipocyte commitment. Furthermore, exposure of A33 cells to noggin, a naturally occurring BMP-4-binding antagonist, during this critical time window blocks subsequent differentiation. The role of BMP-4 in adipocyte lineage commitment is further strengthened by gene expression profiling of proliferating 10T1/2 stem cells and A33 preadipocytes. These findings revealed changes in the molecular circuitry, specifically coordinated changes in the expression of members of the BMP-4 signaling pathway, that distinguish A33 preadipocytes from uncommitted parental 10T1/2 stem cells. Together, these studies provide compelling evidence for the participation of BMP-4 in adipocyte lineage determination.

Adipocytes↗

PubMed2005

Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis.

CCAAT enhancer-binding protein (C/EBP)beta, C/EBPalpha, and peroxisome proliferator activated receptor (PPAR)gamma act in a cascade where C/EBPbeta activates expression of C/EBPalpha and PPARgamma, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C/EBPbeta is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3beta enters the nucleus, which correlates with hyperphosphorylation of C/EBPbeta and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C/EBPbeta show that phosphorylation of Thr-188 by mitogen-activating protein kinase "primes" C/EBPbeta for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3beta, acquisition of DNA-binding function, and transactivation of the C/EBPalpha and PPARgamma genes. The delayed transactivation of the C/EBPalpha and PPARgamma genes by C/EBPbeta appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C/EBPalpha and PPARgamma are antimitotic.

3T3-L1 Cells↗

PubMed2004

Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage.

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1/2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1/2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1/2 stem cells to the adipocyte lineage.

3T3-L1 Cells↗

PubMed2003

Convergence of peroxisome proliferator-activated receptor gamma and Foxo1 signaling pathways.

The forkhead factor Foxo1 (or FKHR) was identified in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor (PPAR) gamma-interacting protein. Foxo1 antagonized PPARgamma activity and vice versa indicating that these transcription factors functionally interact in a reciprocal antagonistic manner. One mechanism by which Foxo1 antagonizes PPARgamma activity is through disruption of DNA binding as Foxo1 inhibited the DNA binding activity of a PPARgamma/retinoid X receptor alpha heterodimeric complex. The Caenorhabditis elegans nuclear hormone receptor, DAF-12, interacted with the C. elegans forkhead factor, DAF-16, paralleling the interaction between PPARgamma and Foxo1. daf-12 and daf-16 have been implicated in C. elegans insulin-like signaling pathways, and PPARgamma and Foxo1 likewise have been linked to mammalian insulin signaling pathways. These results suggest a convergence of PPARgamma and Foxo1 signaling that may play a role in insulin action and the insulinomimetic properties of PPARgamma ligands. A more general convergence of nuclear hormone receptor and forkhead factor pathways may be important for multiple biological processes and this convergence may be evolutionarily conserved.

3T3-L1 Cells↗

PubMed2003

CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis.

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAATenhancer-binding protein (CEBP)beta is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, CEBPbeta activates expression of CEBPalpha and peroxisome proliferator-activated receptor gamma, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. CEBPbeta is expressed immediately after induction but exhibits delayed acquisition of DNA-binding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from CEBPbeta(-/-) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of CEBPbeta (LAP) but not dominant-negative CEBPbeta (LIP) in CEBPbeta(-/-) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of CEBPbeta is a prerequisite for MCE in the adipocyte-differentiation program.

3T3 Cells↗

PubMed2002

Mitotic clonal expansion: a synchronous process required for adipogenesis.

When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G(1)S checkpoint synchronously as evidenced by the expressionactivation of cdk2-cyclin-EA, turnover of p27kip1, hyperphosphorylation of Rb, translocation of cyclin D(1) from nuclei to cytoplasm and GSK-3beta from cytoplasm to nuclei, and incorporation of [(3)H]thymidine into DNA. As the cells cross the G(1)S checkpoint, CEBPbeta acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinaseextracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.

3T3 Cells↗

PubMedDate not supplied

Adipose development: from stem cell to adipocyte.

Cell culture models have been developed to study commitment and subsequent differentiation of preadipocytes into adipocytes. Bone morphogenetic protein 4 commits mesenchymal stem cells to the adipose lineage. Other factors, including Wnt signaling, cell density, and cell shape, play a role in lineage commitment. Following commitment to the adipose lineage, growth-arrested preadipocytes can differentiate to adipocytes by treatment with insulin-like growth factor 1, glucocorticoid and an agent that increases cAMP level. This process is characterized by a rapid and transient increase in CCAAT/enhancer binding protein (C/EBP) beta and synchronous re-entry into the cell cycle. Acquisition of DNA-binding by C/EBPbeta occurs after the transcription factor becomes phosphorylated. The cells enter a growth-arrested state and begin terminal differentiation. C/EBPalpha, peroxisome proliferator-activated receptor gamma, and adipocyte determination, and differentiation-dependent factor 1 coordinate the expression of genes that create and maintain the adipocyte phenotype.

Adipocytes↗