The 5′-AT-rich half-site of Maf recognition element: a functional target for bZIP transcription factor Maf

The Maf family of proteins are a subgroup of basic region-leucine zipper (bZIP) transcription factors, which recognize a long palindromic DNA sequence [TGCTGAC(G)TCAGCA] known as the Maf recognition element (MARE). Interestingly, the functional target enhancer sequences present in the αA-crystallin gene contain a well-conserved half-site of MARE rather than the entire palindromic sequence. To resolve how Maf proteins bind to target sequences containing only MARE half-sites, we examined their binding activities using electrophoretic gel mobility shift assays as well as in vitro and in vivo reporter assays. Our results indicate that the 5′-flanking region of the MARE half-site is required for Maf proteins to bind both in vitro and in vivo. The critical 5′-flanking sequences for c-Maf were determined by a selection and amplification binding assay and show a preference for AT-rich nucleotides. Furthermore, sequence analysis of the regulatory regions of several target genes also suggests that AT-rich sequences are important. We conclude that Maf can bind to at least two types of target sequences, the classical MARE (palindrome type) and a 5′-AT-rich MARE half-site (half-site type). Our results provide important new insights into the DNA binding and site selection by bZIP transcription factors

Necdin Controls Proliferation of White Adipocyte Progenitor Cells

White adipose tissues are composed mainly of white fat cells (adipocytes), which play a key role in energy storage and metabolism. White adipocytes are terminally differentiated postmitotic cells and arise from their progenitor cells (preadipocytes) or mesenchymal stem cells residing in white adipose tissues. Thus, white adipocyte number is most likely controlled by the rate of preadipocyte proliferation, which may contribute to the etiology of obesity. However, little is known about the molecular mechanisms that regulate preadipocyte proliferation during adipose tissue development. Necdin, which is expressed predominantly in postmitotic neurons, is a pleiotropic protein that possesses anti-mitotic and pro-survival activities. Here we show that necdin functions as an intrinsic regulator of white preadipocyte proliferation in developing adipose tissues. Necdin is expressed in early preadipocytes or mesenchymal stem cells residing in the stromal compartment of white adipose tissues in juvenile mice. Lentivirus-mediated knockdown of endogenous necdin expression in vivo in adipose tissues markedly increases fat mass in juvenile mice fed a high-fat diet until adulthood. Furthermore, necdin-null mutant mice exhibit a greater expansion of adipose tissues due to adipocyte hyperplasia than wild-type mice when fed the high-fat diet during the juvenile and adult periods. Adipose stromal-vascular cells prepared from necdin-null mice differentiate in vitro into a significantly larger number of adipocytes in response to adipogenic inducers than those from wild-type mice. These results suggest that necdin prevents excessive preadipocyte proliferation induced by adipogenic stimulation to control white adipocyte number during adipose tissue development

Necdin is expressed in adipose stromal cells <i>in vivo</i>.

<p>(<i>A</i>) Expression of necdin in WAT stroma. Interscapular WAT sections were prepared from 5-week-old male mice and co-stained with Nile red for mature adipocytes and antibodies against necdin, CD34 and Sca-1. (<i>B</i>) Expression of necdin in CD34⁺ and Sca-1⁺ cells. Arrowheads point to representative double-immunopositive cells. (<i>C</i>) Three-dimensional images of intracellular necdin, CD34 and Sca-1. Immunostained tissues were observed by multiple z-stack confocal laser-scanning microscopy. Accessory panels are along XZ and YZ axes. Scale bars; 20 µm (<i>A</i>), 10 µm (<i>B</i>), 5 µm (<i>C</i>).</p

Necdin is expressed in primary adipose SV cells.

<p>(<i>A</i>) Western blot analysis of endogenous necdin. WAT stromal-vascular fraction (SVF), adipocyte fraction (AF), pooled WAT (WAT), interscapular BAT (BAT), and brain were prepared from 5-week-old mice. Necdin in the extracts was analyzed by Western blotting. Molecular sizes are in kilodaltons (kDa). (<i>B</i>) qRT-PCR for necdin mRNA. Total RNA was extracted from the above fractions and tissues. Necdin mRNA was analyzed by qRT-PCR. (<i>C</i>) Immunocytochemistry. Primary SV cells were prepared from 5-week-old mice, cultured, and stained by immunocytochemistry for necdin (red) and CD34, Sca-1, αSMA, or PDGFRβ (green). Double-stained images are merged with nuclear DNA staining (blue) (Merge). Arrowheads point to cells co-expressing necdin and the marker protein. Scale bar, 20 µm.</p

Necdin deficiency enhances adipocyte differentiation in adipose SV cells.

<p>(<i>A</i>, <i>B</i>) PPARγ⁺ cells. SV cells prepared from <i>Ndn</i>^+/+ and <i>Ndn</i>^+m/−p littermates were treated with adipogenic inducers and double-stained for PPARγ (green) and nuclear DNA (blue)(<i>A</i>). PPARγ⁺ cells (arrowheads in <i>A</i>) were counted (mean ± SEM, <i>n</i> = 3)(<i>B</i>). (<i>C, D</i>) Expression levels of <i>PPARγ2</i> and <i>C/EBPα</i> mRNAs. <i>PPARγ2</i> (<i>C</i>) and <i>C/EBPα</i> (<i>D</i>) mRNA levels were analyzed by qRT-PCR 72 hr after adipogenic induction. (<i>E, F</i>) Oil Red O-staining. SV cells were stained with Oil Red O 8 days after adipogenic induction (<i>E</i>), and intracellular Oil Red O was quantified by spectrophotometry (mean ± SEM, <i>n</i> = 4–6)(<i>F</i>). (<i>G, H</i>) Expression levels of <i>aP2</i> and <i>adiponectin</i> mRNAs. The <i>aP2</i> (<i>G</i>) and <i>adiponectin</i> (<i>H</i>) mRNA levels were analyzed by qRT-PCR 8 days after adipogenic induction (mean ± SEM, <i>n</i> = 3). All mRNA levels (<i>C, D, G, H</i>) are shown as relative values to <i>GAPDH</i> mRNA levels (mean ± SEM, <i>n</i> = 3) *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. Scale bars; 20 µm (<i>A</i>), 100 µm (<i>E</i>).</p

Necdin deficiency enhances preadipocyte proliferation in adipose SV cells.

<p>(<i>A</i>) Co-immunostaining for necdin and PPARγ. SV cells were treated with (+) or without (−) adipogenic inducers, and fixed 72 hr after induction. Cells were double-immunostained for necdin (red) and PPARγ (green), and confocal laser microscopic images are merged (Merge). Arrowheads point to the nucleus. (<i>B</i>) Co-staining for BrdU and PPARγ. SV cells were treated with adipogenic inducers, pulse-labeled with BrdU, triple-stained for BrdU, PPARγ and nuclear DNA with Hoechst 33342, and observed by fluorescence microscopy. Arrowheads (Merge) point to BrdU⁺ PPARγ⁺ cells. (<i>C, D</i>) Flow cytometry for BrdU incorporation. SV cells were prepared from <i>Ndn</i>^+/+ and <i>Ndn</i>^+m/−p mice, treated with adipogenic inducers, and analyzed by flow cytometry for BrdU incorporation. BrdU⁺ cells in the colored area (blue for <i>Ndn</i>^+/+, red for <i>Ndn</i>^+m/−p)(<i>C</i>) were counted (<i>D</i>). The threshold (broken line) was set using negative control cells without BrdU treatment (Ctl). (<i>E, F</i>) Flow cytometry for apoptosis. SV cells were treated with adipogenic inducers and analyzed 20 hr later by flow cytometry using FITC-labeled Annexin V. *<i>P</i><0.05. NS, not significant (<i>P</i>>0.05). Scale bars; 10 µm (<i>A</i>), 20 µm (<i>B</i>).</p

There is no difference in white adipocyte size between <i>Ndn</i>^+/+ and <i>Ndn</i>^+m/−p mice.

<p>(<i>A</i>–<i>C</i>) Adipocyte size analyses. WATs of <i>Ndn</i>^+/+ and <i>Ndn</i>^+m/−p littermates fed the high-fat diets from 5 to 14 weeks of age were embedded in paraffin. The WAT sections of 5 µm thickness were stained with hematoxylin and eosin (<i>A</i>). Adipocyte diameters in interscapular (isWAT) and epididymal WATs (epWAT) were measured using NIH Image J software, and shown as frequency distribution (<i>B</i>) and mean diameter ± SEM (<i>C</i>)(>400 cells per section, <i>n</i> = 3 for <i>Ndn</i>^+/+, <i>n</i> = 4 for <i>Ndn</i>^+m/−p). Scale bar, 100 µm. (<i>D</i>) Densities of adipocytes in WATs. Adipocyte densities in the stained sections (observed areas, 300–600 µm²) are presented as the number of adipocytes per square millimeter (examined 3 non-overlapping areas per slice, mean ± SEM, <i>n</i> = 3). NS, not significant (<i>P</i>>0.05) (<i>C</i>, <i>D</i>).</p

Lentivirus-mediated necdin knockdown enhances WAT expansion <i>in vivo</i>.

<p>(<i>A</i>) Representative mice infected with lentivirus vectors for control scrambled RNA (cRNA) and necdin shRNA (shRNA). Mice were infected with the recombinant lentiviruses at 5 weeks of age and subsequently fed a high-fat diet for 6 weeks. Red arrowheads point to the injection sites in the interscapular WAT. (<i>B</i>, <i>C</i>) Representative interscapular (<i>B</i>) and epididymal fat pads (<i>C</i>). WATs were excised from lentivirus-infected mice. Arrowheads point to brown fat depots (<i>B</i>) and testes (<i>C</i>) attached to the fat pads for orientation. Scale bars, 1 cm. (<i>D</i>, <i>E</i>) White fat pad weight. Interscapular (<i>D</i>) and epididymal (<i>E</i>) white fat pads excised from lentivirus-infected mice fed the high-fat diet were weighed after removing non-white-adipose tissues. Absolute (left) and relative values (right) are shown (mean ± SEM, <i>n</i> = 4). **<i>P</i><0.01, ***<i>P</i><0.001.</p