Summary
Nutritional and pharmacological stimuli can dramatically alter the cellular phenotypes in white adipose tissue (WAT). Utilizing genetic lineage tracing techniques, we demonstrate that brown adipocytes (BA) that are induced by β3-adrenergic receptor activation in abdominal WAT arise from the proliferation and differentiation of cells expressing platelet-derived growth factor receptor alpha (PDGFRα), CD34 and Sca1 (PDGFRα+ cells). PDGFRα+ cells have a unique morphology in which extended processes contact multiple cells in the tissue microenvironment. Surprisingly, these cells also give rise to white adipocytes (WA) that can comprise up to 25% of total fat cells in abdominal fat pads following 8 weeks of high fat feeding. Isolated PDGFRα+ cells differentiated into both BA and white adipocytes (WA) in vitro, and generated WA after transplantation in vivo. The identification of PDGFRα+ cells as bipotential adipocyte progenitors will enable further investigation of mechanisms that promote therapeutic cellular remodeling in adult WAT.
SUMMARY
The regulatory events guiding progenitor activation and differentiation in adult white adipose tissue are largely unknown. We report that induction of brown adipogenesis by β3-adrenergic receptor (ADRB3) activation involves the death of white adipocytes and their removal by M2-polarized macrophages. Recruited macrophages express high levels of osteopontin (OPN), which attracts a subpopulation of PDGFRα+ progenitors expressing CD44, a receptor for OPN. Preadipocyte proliferation is highly targeted to sites of adipocyte clearance and occurs almost exclusively in the PDGFRα+ CD44+ subpopulation. Knockout of OPN prevents formation of crown-like structures by ADRB3 activation and the recruitment, proliferation, and differentiation of preadipocytes. The recruitment and differentiation of PDGFRα+ progenitors are also observed following physical injury, during matrix-induced neogenesis, and in response to high-fat feeding. Each of these conditions recruits macrophages having a unique polarization signature, which may explain the timing of progenitor activation and the fate of these cells in vivo.
This work investigated how cold stress induces the appearance of brown adipocytes (BAs) in brown and white adipose tissues (WATs) of adult mice. In interscapular brown adipose tissue (iBAT), cold exposure increased proliferation of endothelial cells and interstitial cells expressing platelet-derived growth factor receptor, α polypeptide (PDGFRα) by 3- to 4-fold. Surprisingly, brown adipogenesis and angiogenesis were largely restricted to the dorsal edge of iBAT. Although cold stress did not increase proliferation in inguinal white adipose tissue (ingWAT), the percentage of BAs, defined as multilocular adipocytes that express uncoupling protein 1, rose from undetectable to 30% of total adipocytes. To trace the origins of cold-induced BAs, we genetically tagged PDGFRα(+) cells and adipocytes prior to cold exposure, using Pdgfra-Cre recombinase estrogen receptor T2 fusion protein (CreER(T2)) and adiponectin-CreER(T2), respectively. In iBAT, cold stress triggered the proliferation and differentiation of PDGFRα(+) cells into BAs. In contrast, all newly observed BAs in ingWAT (5207 out of 5207) were derived from unilocular adipocytes tagged by adiponectin-CreER(T2)-mediated recombination. Surgical denervation of iBAT reduced cold-induced brown adipogenesis by >85%, whereas infusion of norepinephrine (NE) mimicked the effects of cold in warm-adapted mice. NE-induced de novo brown adipogenesis in iBAT was eliminated in mice lacking β1-adrenergic receptors. These observations identify a novel tissue niche for brown adipogenesis in iBAT and further define depot-specific mechanisms of BA recruitment.
Heparin-binding EGF-like growth factor (HBEGF), which is expressed in the placenta during normal pregnancy, is downregulated in pre-eclampsia, a human pregnancy disorder associated with poor trophoblast differentiation and survival. This growth factor protects against apoptosis during stress, suggesting a role in trophoblast survival in the relatively low O 2 (~2%) environment of the first trimester conceptus. Using a well-characterized human first trimester cytotrophoblast cell line, we found that a 4-hour exposure to 2% O 2 upregulates HBEGF synthesis and secretion independently of an increase in its mRNA. Five other expressed members of the EGF family are largely unaffected. At 2% O 2 , signaling via HER1 or HER4, known HBEGF receptors, is required for both HBEGF upregulation and protection against apoptosis. This positive-feedback loop is dependent on metalloproteinasemediated cleavage and shedding of the HBEGF ectodomain. The restoration of trophoblast survival by the addition of soluble HBEGF in cultures exposed to low O 2 and metalloproteinase inhibitor suggests that the effects of HBEGF are mediated by autocrine/paracrine, rather than juxtacrine, signaling. Our results provide evidence that a post-transcriptional mechanism induced in trophoblasts by low O 2 rapidly amplifies HBEGF signaling to inhibit apoptosis. These findings have a high clinical significance, as the downregulation of HBEGF in pre-eclampsia is likely to be a contributing factor leading to the demise of trophoblasts.
Objective-The anti-apoptotic action of HBEGF and its regulation by O 2 constitutes a key factor for trophoblast survival. The hypothesis that cytotrophoblast survival is compromised by exposure to hypoxia-reoxygenation (H/R) injury, which may contribute to preeclampsia and some missed abortions, prompted us to investigate HBEGF regulation and its role as a survival factor during H/ R in cytotrophoblast cells Study Design-A transformed human first trimester cytotrophoblast cell line HTR-8/SVneo was exposed to H/R (2% O 2 followed by 20% O 2 ) and assessed for HBEGF expression and cell death.Results-Cellular HBEGF declined significantly within 30 minutes of reoxygenation after culture at 2% O 2 . H/R significantly reduced proliferation and increased cell death when compared to trophoblast cells cultured continuously at 2% or 20% O 2 . Restoration of cell survival also was achieved by adding recombinant HBEGF during reoxygenation. HBEGF inhibited apoptosis through its binding to either HER1 or HER4, its cognate receptors.Conclusion-These results provide evidence that cytotrophoblast exposure to H/R induces apoptosis and decreased cell proliferation. HBEGF accumulation is diminished under these conditions, while restoration of HBEGF signaling improves trophoblast survival.
Heparin-binding EGF-like growth factor (HBEGF) induces trophoblast extravillous differentiation and prevents apoptosis. These functions are compromised in preeclampsia. Because HBEGF is downregulated in placentas delivered by women with preeclampsia, we have examined its expression and cytoprotective activity in term villous explants. Chorionic villous explants prepared from nonpathological placentas collected by cesarean section at term were cultured at either 20% or 2% O 2 and treated with the HBEGF antagonist CRM197 or recombinant HBEGF. Paraffin sections were assayed for trophoblast death, proliferation and HBEGF expression using the TUNEL method, immunohistochemistry for nuclear Ki67 expression and semi-quantitative immunohistochemistry with image analysis, respectively. Trophoblast cell death was increased significantly after 8 h of culture with CRM197 or by culture for 2 h at 2% O 2 . Exogenous HBEGF prevented cell death due to hypoxia. Proliferative capacity was not affected by culture at either 20% or 2% O 2 . Contrary to first trimester placenta, term trophoblasts do not elevate HBEGF expression in response to hypoxia. However, low endogenous levels of HBEGF are required to maintain survival. Therefore, HBEGFmediated signaling significantly reduces trophoblast cell death at term and its deficiency in preeclampsia could negatively impact trophoblast survival.
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