Cellular differentiation involves transcriptional responses to environmental stimuli. Adipocyte differentiation is inhibited under hypoxic conditions, indicating that oxygen (O(2)) is an important physiological regulator of adipogenesis. Hypoxia inhibits PPAR gamma 2 nuclear hormone receptor transcription, and overexpression of PPAR gamma 2 or C/EBP beta stimulates adipogenesis under hypoxia. Mouse embryonic fibroblasts deficient in hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) are refractory to hypoxia-mediated inhibition of adipogenesis. The HIF-1-regulated gene DEC1/Stra13, a member of the Drosophila hairy/Enhancer of split transcription repressor family, represses PPAR gamma 2 promoter activation and functions as an effector of hypoxia-mediated inhibition of adipogenesis. These data indicate that an O(2)-sensitive signaling mechanism regulates adipogenesis. Thus, agents that regulate HIF-1 activity or O(2) sensing may be used to inhibit adipogenesis and control obesity.

Summary MicroRNAs (miRNAs) are involved in a plethora of important biological processes from embryonic development to homeostasis in adult tissues. Recently, miRNAs have emerged as a class of epigenetic regulators of metabolism and energy homoestasis. We have investigated the role of miRNAs in the regulation of adipogenic differentiation. In this report, we demonstrate that the miR-27 gene family is down-regulated during adipogenic differentiation. Over-expression of miR-27 specifically inhibits adipocyte formation, without affecting myogenic differentiation. We also find that expression of miR-27 results in blockade of expression of PPARγ and C/EBPα, the two master regulators of adipogenesis. Importantly, expression of miR-27 is increased in fat tissue of obese mice and is regulated by hypoxia, an important extracellular stress associated with obesity. Our data strongly suggest that miR-27 is a new class of adipogenic inhibitors and may play a role in the pathological development of obesity.

Previous studies have indicated that myoblasts can differentiate and repair muscle injury after an ischemic insult. However, it is unclear how hypoxia or glucose deprivation in the ischemic microenvironment affects myoblast differentiation. We have found that myogenesis can adapt to hypoxic conditions. This adaptive mechanism is accompanied by initial inhibition of the myoD, E2A, and myogenin genes followed by resumption of their expression in an oxygen-dependent manner. The regulation of myoD transcription by hypoxia is correlated with transient deacetylation of histones associated with the myoD promoter. It is noteworthy that, unlike the differentiation of other cell types such as preadipocytes or chondroblasts, the effect of hypoxia on myogenesis is independent of HIF-1, a ubiquitous regulator of transcription under hypoxia. While myogenesis can also adapt to glucose deprivation, the combination of severe hypoxia and glucose deprivation found in an ischemic environment results in pronounced loss of myoblasts. Our studies indicate that the ischemic muscle can be repaired via the adaptive differentiation of myogenic precursors, which depends on the levels of oxygen and glucose in the ischemic microenvironment.Skeletal muscle possesses the remarkable ability to withstand chronic ischemia. However, the newly developed muscle fibers tend to be smaller in diameter than those of healthy, nonischemic myofibers (46). Interestingly, chronic exposure to high altitude can also result in smaller myofiber area and lower muscle mass in mountaineers (25). Similar myofiber differences are also observed in healthy people who live at high altitude for generations (25). Taken together, these observations suggest that decreased oxygen tension or hypoxia alone may be sufficient to alter the differentiation of myoblasts.Ischemic muscle damage is repaired by myogenic satellite cells, a small population of precursor cells located between myofibers (12,23,47). When stimulated, the otherwise quiescent satellite cells undergo terminal differentiation into myocytes to regenerate damaged myofibers. It has been found that cultured satellite cells form new muscle fibers when transplanted into ischemic muscle (12). The myogenic differentiation of myoblasts is highly orchestrated by a family of myogenic regulatory factors (MRFs), such as myoD, myogenin, myf5, and MRF4 in collaboration with the E2A gene products (E12 or E47) and/or the myogenic enhancing factors (6, 41, 53). Although a plethora of information on the embryonic development of muscle exists, there is still a lack of clear understanding of how postnatal myogenic satellite cells are regulated during muscle regeneration, especially by their microenvironment.Tissue ischemia in myocardium and skeletal muscle results in hypoxia and elevates the expression of the hypoxia-inducible transcription factor HIF-1␣ and its downstream gene, VEGF (32,40). Although ischemic tissue is exposed to hypoxia and decreased nutritional supply, cells will alter their metabolism, proliferation, and differe...

The stem cell niche is a unique tissue microenvironment that regulates the self-renewal and differentiation of stem cells. Although several stromal cells and molecular pathways have been identified, the microenvironment of the stem cell niche remains largely unclear. Recent evidence suggests that stem cells are localized in areas with low oxygen. We have hypothesized that hypoxia maintains the undifferentiated phenotype of stem/precursor cells. In this report, we demonstrate that hypoxia reversibly arrests preadipocytes in an undifferentiated state. Consistent with this observation, hypoxia maintains the expression of pref-1, a key stem/precursor cell gene that negatively regulates adipogenic differentiation. We further demonstrate that the hypoxia-inducible factor-1 (HIF-1) constitutes an important mechanism for the inhibition of adipogenic differentiation by hypoxia. Our findings suggest that hypoxia in the stem cell niche is critical for the maintenance of the undifferentiated stem or precursor cell phenotype.Stem and/or precursor cells exist in a distinct tissue structure called the niche that regulates the self-renewal and differentiation of stem cells (1, 2). As shown recently, the bone marrow microenvironment has lower oxygen concentration than other tissues and stem cells are localized in the hypoxic regions (3), suggesting that hypoxia may be important for stem cell maintenance. However, the role of hypoxia in stem cell maintenance remains to be fully understood.Hypoxia can regulate cellular differentiation. Under hypoxic conditions, the differentiation of embryonal stem cells, as well as precursor cells is inhibited (4 -6). Studies in cancer biology have shown that hypoxia is strongly correlated with an undifferentiated phenotype in solid tumors such as neuroblastoma (7), breast cancer (8), and cervical cancer (9). These observations indicate that hypoxia plays a critical role in the maintenance of the undifferentiated stem cell phenotype.Cellular response to hypoxia is manifested by the activation of the hypoxia-inducible factor-1 (HIF-1), 4 a transcription factor of the basic helix-loop-helix Per, Arnt, and Sim family (10, 11). HIF-1 consists of the O 2 -regulated HIF-1␣ subunit and the O 2 -independent HIF-1␤ subunit. Under normoxia, HIF-1␣ protein becomes hydroxylated at proline-402 and proline-564 in its O 2 -dependent degradation (ODD) domain and is targeted by the von Hippel-Lindau protein for proteasome-mediated degradation (10, 11). As pO 2 decreases to hypoxic levels, HIF-1␣ is no longer hydroxylated and thus becomes stabilized. Upon nuclear translocation, HIF-1␣ dimerizes with the O 2 -independent HIF-1␤ to initiate gene transcription (10, 11). HIF activation results in increased expression of several key stem cell markers such as CXCR4 (12, 13), SDF-1/CCL12 (3), and OCT4 (14). Conversely, the prodifferentiation gene, peroxisome proliferator-activated receptor ␥ (PPAR␥), is down-regulated as a result of HIF activation (6). Using the 3T3-L1 preadipocytes as a model, we have investigated the e...

Reduced oxygenation, or hypoxia, inhibits differentiation and facilitates stem cell maintenance. Hypoxia commonly occurs in solid tumors and promotes malignant progression. Hypoxic tumors are aggressive and exhibit stem celllike characteristics. It remains unclear, however, whether and how hypoxia regulates cancer cell differentiation and maintains cancer cell stemness. Here, we show that hypoxia increases the expression of the stem cell gene DLK1, or delta-like 1 homologue (Drosophila), in neuronal tumor cells. Inhibition of DLK1 enhances spontaneous differentiation, decreases clonogenicity, and reduces in vivo tumor growth. Overexpression of DLK1 inhibits differentiation and enhances tumorigenic potentials. We further show that the DLK1 cytoplasmic domain, especially Tyrosine339 and Serine355, is required for maintaining both clonogenicity and tumorigenicity. Because elevated DLK1 expression is found in many tumor types, our observations suggest that hypoxia and DLK1 may constitute an important stem cell pathway for the regulation of cancer stem cell-like functionality and tumorigenicity. [Cancer Res 2009;69(24):9271-80]

The transcription factor HIF-1α activity is increased in adipose tissue to contribute to chronic inflammation in obesity. However, its upstream and downstream events remain to be characterized in adipose tissue in obesity. We addressed this issue by investigating adipocyte HIF-1α activity in response to obesity-associated factors, such as adipogenesis, insulin, and hypoxia. In adipose tissue, both HIF-1α mRNA and protein were increased by obesity. The underlying mechanism was investigated in 3T3-L1 adipocytes. HIF-1α mRNA and protein were augmented by adipocyte differentiation. In differentiated adipocytes, insulin further enhanced HIF-1α in both levels. Hypoxia enhanced only HIF-1α protein, not mRNA. PI3K and mTOR activities are required for the HIF-1α expression. Function of HIF-1α protein was investigated in the regulation of VEGF gene transcription. ChIP assay shows that HIF-1α binds to the proximal hypoxia response element in the VEGF gene promoter, and its function is inhibited by a corepressor composed of HDAC3 and SMRT. These observations suggest that of the three obesity-associated factors, all of them are able to augment HIF-1α protein levels, but only two (adipogenesis and insulin) are able to enhance HIF-1α mRNA activity. Adipose tissue HIF-1α activity is influenced by multiple signals, including adipogenesis, insulin, and hypoxia in obesity. The transcriptional activity of HIF-1α is inhibited by HDAC3-SMRT corepressor in the VEGF gene promoter.

Hypoxia or oxygen deficiency is a salient feature of solid tumors. Hypoxic tumors are often resistant to conventional cancer therapies, and tumor hypoxia correlates with advanced stages of malignancy. Hypoxic tumors appear to be poorly differentiated. Increasing evidence suggests that hypoxia has the potential to inhibit tumor cell differentiation and thus plays a direct role in the maintenance of cancer stem cells. Studies have also shown that hypoxia blocks differentiation of mesenchymal stem/progenitor cells, a potential source of tumor-associated stromal cells. It is therefore likely that hypoxia may have a profound impact on the evolution of the tumor stromal microenvironment. These observations have led to the emergence of a novel paradigm for a role of hypoxia in facilitating tumor progression. Hypoxia may help create a microenvironment enriched in poorly differentiated tumor cells and undifferentiated stromal cells. Such an undifferentiated hypoxic microenvironment may provide essential cellular interactions and environmental signals for the preferential maintenance of cancer stem cells. This hypothesis suggests that effectively targeting hypoxic cancer stem cells is a key to successful tumor control.

The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin–mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519–527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti–C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.