Stem Cells, Vol. 15, No. 5, 334-339,
September 1997
© 1997 AlphaMed Press
A Novel Gene (drad-1) Expressed in Hematopoiesis-Supporting Stromal Cell Lines, ST2, PA6 and A54 Preadipocytes: Use of mRNA Differential Display
Takami L. Maekawaa,
Tsuneo A. Takahashia,
Mitsuhiro Fujiharaa,
Noriko Urushibaraa,
Emi Kadowaki-Kikuchia,
Mitsuo Nishikawab,
Kenji Ikebuchia,
Shigetaka Asanob,
Keiya Ozawac,
Sadayoshi Sekiguchia
a Japanese Red Cross, Hokkaido Red Cross Blood Center, Sapporo, Japan;
b The Department of Hematology-Oncology and Blood Transfusion, The Institute of Medical Science, The University of Tokyo;
c Department of Molecular Biology, Institute of Hematology, Jichi Medical School, Tochigi, Japan
Key Words. Hematopoiesis • Stromal cell • C3H10T1/2 • Differentiation • Preadipocyte • Adipocyte
Correspondence:
Dr. Takami L. Maekawa, Hokkaido Red Cross Blood Center, Yamanote 2-2, Nishi-ku, Sapporo 063, Japan.
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Abstract
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We established a differentiation-inducible preadipocyte cell line, designated A54 preadipocytes, from C3H10T1/2 (10T1/2) mouse embryo fibroblasts. A54 preadipocytes had marked hematopoiesis-supporting ability in vitro but this ability was lost after terminal differentiation to adipocytes. In this study, to identify molecules that contribute to the hematopoiesis-supporting ability of A54 preadipocytes, we screened genes that were differentially expressed in A54 preadipocytes and isolated seven novel genes by reverse transcriptase polymerase chain reaction mRNA differential display. An RNase protection assay confirmed that one of these genes was expressed at high levels in parent 10T1/2 cells and A54 preadipocytes but to a much lesser extent in fully differentiated A54 adipocytes. This gene was defined as a gene that was downregulated during adipocyte differentiation-1 (drad-1). The size of drad-1 mRNA was 8.2 kb, and the gene was expressed in other mouse preadipocytes, namely, ST2 and PA6 cells, that have hematopoiesis-supporting ability. Moreover, drad-1 was also found to be expressed in bone marrow in vivo. The function of the protein encoded by drad-1 is unknown, but the expression of the gene may be useful as a molecular marker of adipocyte differentiation.
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Introduction
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It is difficult to enhance the self-renewal or expansion of pluripotent hematopoietic stem cells and/or immature progenitor cells in vitro using exogenous cytokines [1-4]. However, it is possible to maintain for long term or to support the growth of immature hematopoietic progenitor cells by coculture with stromal cells [5-11]. The cited studies suggest that the stromal cells succeed in constructing some kind of hematopoiesis-inductive environment in vitro by producing hematopoiesis-regulating factors. Thus, we might expect that the factors produced by stromal cells would not only include well-known cytokines but would also include unknown factors, since it is difficult to reconstruct a hematopoiesis-supporting environment using only known exogenous cytokines and other materials.
We previously established a cell line, namely, A54 preadipocytes, from 10T1/2 mouse embryo fibroblasts [10], that differentiated into adipocytes (A54 adipocytes) under appropriate conditions. Sorted progenitor cells were cocultured with 10T1/2-derived cell lines, with subsequent analysis of the number of colony-forming units granulocyte-macrophage (CFU-GM) before and after seven days of the coculture. The number of CFU-GM was maintained or increased in cocultures with parent 10T1/2 cells or A54 preadipocytes in the absence of additional cytokines. By contrast, mature A54 adipocytes did not support growth of CFU-GM [10].
The differences between the hematopoiesis-supporting ability of A54 preadipocytes and that of A54 adipocytes might be due to proteins that are differentially expressed in A54 preadipocytes and A54 adipocytes. Moreover, it should be possible to identify the unknown factor(s) that is produced specifically by preadipocytes and which contributes to the hematopoiesis-supporting ability. The use of the A54 preadipocytes/adipocyte system has a great advantage with respect to a search for such stromal factor(s) since the latter cells are derived from the former and, therefore, the differences in gene expression should be limited. We postulated that it should be possible to identify preadipocyte-specific genes by gene subtraction techniques using these cell lines and, indeed, in this study, we identified seven novel genes that were specifically expressed in the preadipocytes by exploiting the mRNA differential display technique [12]. The genes identified in A54 preadipocytes are candidates for genes that might contribute to the hematopoiesis-supporting ability. Among these genes, we characterized one gene in detail.
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Materials and Methods
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Cell Culture and Induction of Differentiation
Establishment of the A54 preadipocytes cell line and induction of the differentiation from preadipocytes to adipocytes were carried out as described previously [10]. A54 preadipocytes were induced to differentiate to A54 adipocytes by supplementing culture medium with 10 µM insulin (Wako Chemicals; Tokyo, Japan) and 1 µM dexamethasone (Sigma; St. Louis, MO). After culture for seven days, approximately 97% of cells were judged to be terminally differentiated adipocytes from observations of fat droplets in the cells. ST2 and PA6 cells were cultured as described in [5] and [6], respectively. DA-1 cells were cultured in IMDM medium containing 10% fetal calf serum (FCS) and 5 ng/ml mouse interleukin 3 (IL-3) (Kirin Brewery Co., Ltd.; Tokyo, Japan). RAW264.7 cells were cultured in DMEM medium containing 10% FCS. Cells were cultured at 37°C in an incubator with 5% CO2.
Preparation of RNA
All stromal cell lines were cultured until cells reached confluence, and the cells were stimulated by addition of 10 µg/ml lipopolysaccharide ([LPS]; Sigma) to the culture medium. After a 15-h incubation, total RNA was extracted from the cells by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method [13]. Messenger RNA was prepared from LPS-treated A54 preadipocytes with a FastTrack mRNA isolation kit (Invitrogen Corp.; San Diego, CA). Bone marrow, spleen, and thymus cells that were obtained from the femurs of C57BL/6 mice (Sankyo Labo Service Corp., Inc.; Tokyo, Japan) were washed once in IMDM/10% FCS, and then RNA was extracted by the AGPC method.
Analysis of Gene Expression by mRNA Differential Display
mRNA differential display was performed as described [12, 14] with an RNAmap kit (GenHunter Corp.; Brookline, MA). Reverse transcription reactions were carried out with 0.2 µg of DNase-I-treated total RNA that had been prepared from A54 preadipocytes, A54 adipocytes and PA6 cells. Amplifications by the polymerase chain reaction (PCR) were carried out with 20 different primer combinations. The products of PCR, labeled with [
-35S]-dATP (Amersham International plc; Buckinghamshire, UK), were subjected to electrophoresis for autoradiography to identify bands that had been differentially amplified from preadipocytes (A54 preadipocytes, PA6 cells) and A54 adipocytes. The bands were extracted from the gel, reamplified, and subcloned into a pCR II vector (Invitrogen Corp.).
RNase Protection Assay
Preparation of the RNA probe and the RNase protection assay were performed with MAXIscript (In vitro transcription kit; Ambion Inc.; Austin, TX) and RPA II (Ribonuclease protection assay kit; Ambion Inc.), respectively. In brief, pCR II vector DNA with the subcloned fragment of the drad-1 gene was linearized by digestion with restriction enzyme and used as template DNA for transcription by T7 RNA polymerase with [-32P]-UTP (Amersham). Ten µg of total RNA prepared from cells and tissues indicated in the text were mixed with the 32P-labeled probe. Then each mixture was incubated at 42 °C for 12 h to permit hybridization of the probe to the drad-1 mRNA in the samples, and the mixture was treated with RNase A and RNase T1 to digest unhybridized RNA. After recovery of undigested RNA by ethanol precipitation, the samples were subjected to electrophoresis and analyzed. For semiquantitative analysis of amounts of transcripts, we compared intensities of bands on an x-ray film. Images of the bands on the film were transported into a computer via a scanner and analyzed with the Gel Plotting Macros program (NIH image, ver. 1.52; National Institutes of Health; Bethesda, MD).
Detection of drad-1 in Mouse Bone Marrow by Nested PCR
Sequences of PCR primers used for amplification of the drad-1 fragment were as follows: primer 1, ACC CTC CCC AAC AAA GCA TG; primer 2, CAC TGA ACT GTG GCC TCC TT; and primer 3, TGT TGT TCT GTT GGC TGA CC. Template cDNA used for PCR was transcribed from mouse bone marrow RNA by reverse transcriptase with an oligo(dT) primer. The first PCR was performed with primer 1 and primer 3 for 30 cycles (94°C for 30 sec, 50°C for 2 min. 72°C for 45 sec). After dilution of the first product (final, 1:500), the second nested PCR was performed with primer 1 and primer 2 for 18 cycles (94°C for 30 sec, 50°C for 2 min, 72°C for 45 sec). The expected size of the final product after the second PCR was 192 bp, and the fragments generated by cleavage at an internal Msc I site were expected to be 69 bp and 123 bp in length.
Other Techniques
Northern analysis was performed as described elsewhere [15]. Preparation of plasmid DNA and sequence analysis of double-stranded DNA were also carried out as described elsewhere [16], and resultant nucleotide sequences were compared with known sequences by searching the GenBank database (version 95) with the FASTA program (DNA Data Bank of Japan; Mishima, Japan).
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Results
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Cloning of Differentiation-Specific Genes
To identify genes that are transcriptionally activated in preadipocytes with hematopoiesis-supporting ability, we compared mRNA differential display patterns between preadipocytes and adipocytes. We carried out amplifications by PCR with 20 different primer combinations for A54 preadipocytes, A54 adipocytes and PA6 cells. About 2,500 amplified fragments of DNA were visualized on a gel from each RNA pool. Among these PCR products, eight bands were identified as preadipocyte-specific bands. Figure 1 shows part of an electrophoresis and autoradiogram image of the PCR products. Bands in lanes 1 to 4 (arrowhead) were specifically amplified reproducibly from RNA of A54 preadipocytes and PA6 cells, although faint bands were amplified from the RNA of A54 adipocytes in lanes 5 and 6. This result suggests that the gene that encoded the amplified fragment was differentially expressed in A54 preadipocytes and PA6 cells. The product of PCR (Fig.1 arrowhead) was a fragment of drad-1 as described in detail below. After subcloning of the eight differentially displayed fragments, including the fragments pointed by the arrowhead in Figure 1, sequence analysis and a search for homologous sequences in GenBank were performed. We found that one PCR product was homologous to a porcine gene for destrin, an actin-binding protein [17]. No sequences similar to the other seven PCR products were found in GenBank, indicating that they represented novel genes.
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Figure 1. Differential display for comparison of RNAs from PA6 cells, A54 preadipocytes and A54 adipocytes. Total RNA was extracted from the indicated cells. Two RT-PCR experiments were performed independently, and the amplified 35S-labeled PCR products were analyzed on a single gel. Odd-numbered and even-numbered lanes represent experiments 1 and 2, respectively. Fragments were reproducibly amplified from PA6 and A54 preadipocyte RNAs in both independent experiments (arrowhead, lanes 1 to 4), whereas the fragment was not amplified from A54 adipocyte RNA (lanes 5 and 6). The fragment was cut out from the gel and subcloned for further analysis.
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Further characterization of one of the seven novel genes, designated drad-1, was performed as described below. The partial nucleic acid sequence of drad-1 (314 bp) will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the accession number D67077.
Northern Blot Analysis of drad-1 mRNA Expression in the A54-preadipocyte
We examined the size of the drad-1 mRNA by Northern blotting. mRNA from A54 preadipocytes was purified, subjected to electrophoresis, and blotted for Northern analysis. Figure 2A shows a blot after hybridization with a 32P-labeled fragment of drad-1 . The size of the major drad-1 transcript was approximately 8.2 kb and that of a minor transcript was approximately 4.6 kb.
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Figure 2. Expression of differentiation-associated genes in A54 preadipocytes and A54 adipocytes. A) 2 µg of mRNA prepared from A54 preadipocytes were used for Northern blot analysis with a 32P-labeled drad-1 gene fragment as probe. An 8.2-kb major band was observed. B) Ethidium bromide-stained image after electrophoresis of 10 µg of total RNA prepared from A54 preadipocytes and A54 adipocytes. C) Northern blot analysis of the expression of adipsin mRNA in A54 preadipocytes and A54 adipocytes. Total RNA was blotted onto a nylon membrane and hybridized with the probe for adipsin mRNA. Adipsin was specifically expressed in A54 adipocytes. D) Expression of drad-1 in A54 preadipocytes and A54 adipocytes, as analyzed by an RNase protection assay. Ten µg of the same RNA as shown in (B) were used as substrate for hybridization with the labeled drad-1 probe. drad-1 was specifically expressed in A54 preadipocytes.
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Expression of drad-1 During Differentiation of Preadipocytes to Adipocytes
We compared levels of expression of drad-1 during differentiation of A54 preadipocytes to A54 adipocytes by an RNase protection assay. Figure 2B shows an ethidium bromide-stained image of a gel after fractionation of 10 µg of total RNA from A54 preadipocytes and A54 adipocytes. It is clear that the preparation of RNA was successful in each case and that almost identical amounts of RNA had been applied to the gel for electrophoresis. Northern analysis ( Fig. 2C) revealed that the mRNA for adipsin, an adipocyte-specific proteinase, was specifically expressed in A54 adipocytes (right lane) but not in A54 preadipocytes (left lane), an indication that induction of the differentiation of preadipocytes to adipocytes had been accomplished successfully.
Then we compared levels of expression of drad-1 between A54 preadipocytes and A54 adipocytes by the RNase protection assay ( Fig. 2D) with the same RNA fractions as had been used for the Northern analysis of adipsin mRNA. After hybridization of the 32P-labeled drad-1 probe to 10 µg of total RNA prepared from A54 preadipocytes and from A54 adipocytes, the unhybridized probe was digested by treatment with RNase. Then the undigested probe was analyzed by electrophoresis. As shown in Figure 2D, an intense band was observed in the case of the A54 preadipocyte fraction (left lane), and a faint band was seen in that of the A54 adipocyte fraction (right lane). This result indicates that drad-1 was expressed in A54 preadipocytes specifically, whereas the level of expression of drad-1 was dramatically decreased in A54 adipocytes. Thus, expression of drad-1 appeared to be regulated in a differentiation-specific manner.
Expression of drad-1 in Other Stromal Cell Lines, Myeloid Cell Lines, and Tissues
We investigated the expression of drad-1 in stromal cell lines, namely, parent 10T1/2, ST2 and PA6. ST2 and PA6 have hematopoiesis-supporting ability. We also investigated drad-1 expression in erythroid DA-1 cells [18], monocyte-macrophage RAW264.7 cells [19], liver, and lymphoid tissues, namely, spleen and thymus ( Fig. 3). Total RNA was prepared from the indicated cells and tissues, and RNase protection assays were performed as described above with the probes for drad-1 and ß-actin. As shown in Figure 3 (A and C, lower panels), mRNA for ß-actin was detected in all preparations of RNA with the exception of yeast RNA (negative controls), indicating that hybridization with the probe and digestion during the RNase protection assay had been carried out successfully. In Figure 3A (upper panel), distinct expression of drad-1 was detected in parent 10T1/2 cells, A54 preadipocytes, ST2 cells, and PA6 cells, while a faint band was observed in the A54 adipocyte fraction. In Figure 3C (lower panel), no band was observed in the DA-1, RAW264.7, spleen, thymus, or liver fraction. To analyze the amount of the drad-1 transcript semiquantitatively, we evaluated the intensities of the bands on the X-ray film with a densitometer, using the intensities of ß-actin bands ( Figs. 3B and 3D). As shown in Figure 3B, the amount of drad-1 mRNA in A54 adipocytes was approximately 5% of that in parent 10T1/2 cells and A54 preadipocytes (columns 1 to 3), indicating that drad-1 was downregulated during differentiation from preadipocytes to adipocytes. Expressed amounts of drad-1 mRNA in ST2 and PA6 cells were similar to those in parent 10T1/2 cells and A54 preadipocytes. drad-1 was not expressed in myeloid cell lines that we examined or in lymphoid cells that were included in spleen or thymus.
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Figure 3. Expression of drad-1 in cell lines and tissues. Total RNA was prepared from indicated cell lines and mouse tissues. Yeast RNAs were used as negative controls. Ten µg of each RNA was allowed to hybridize with the labeled drad-1 probe and digested with RNase. Then the samples were analyzed by electrophoresis and autoradiography. (A and C, upper panels): drad-1 was expressed in parent 10T1/2 cells (A, lane 1) and A54 preadipocytes (A, lane 2). However, the expression of drad-1 was downregulated after differentiation of preadipocytes to adipocytes (A, lane 3). drad-1 was also expressed in ST2 (A, lane 4) and PA6 (A, lane 5 and B, lane 7) cells. No drad-1 expression was observed in DA-1, RAW264.7, spleen, thymus and liver fractions (C, lanes 8 to 12). (A and C, lower panels): Expression of ß-actin mRNA was examined. Expression of neither drad-1 or ß-actin mRNA was detected in the negative controls (yeast RNA; lanes 6 and 13). (B and D): Relative levels of drad-1 transcripts. Columns 1 to 5 correspond to the numbered lanes in panel (A), and columns 7 to 12 correspond to the numbered lanes in panel (C). Expression of drad-1 was evaluated as described in Materials and Methods. Vertical axis: arbitrary units.
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Expression of drad-1 in Bone Marrow
If drad-1 has a functional role in hematopoiesis, the gene should be expressed in bone marrow, a major hematopoietic organ, in adult mice. To examine the expression of drad-1, we extracted total RNA from bone marrow cells from mouse femurs and performed reverse transcription with an oligo(dT) primer. Then nested PCR was performed with drad-1-specific primers, as described in Materials and Methods, and the products were analyzed by agarose gel electrophoresis. As shown in Figure 4 , a positive control produced a fragment of 192 bp (lane 3). A predicted PCR product of 192 bp was also amplified from the mouse bone marrow RNA (lane 2), and the product was cut into two fragments by treatment with Msc I (lane 5). According to the nucleotide sequence of drad-1, a site that is recognized by Msc I should be located in the middle of the PCR product. Negative control PCR, without any template DNA, yielded no amplified bands (lanes 4 and 7). These observations indicate that the amplified PCR product was a fragment of drad-1 and that the gene was expressed in mouse bone marrow.
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Figure 4. Expression of drad-1 in mouse bone marrow. RNA preparation and RT-PCR were performed as described in Materials and Methods. In positive control experiments, a subcloned drad-1 gene fragment was used as template DNA for PCR. Samples were subjected to electrophoresis on a 4% agarose gel. The same-sized products were amplified from the bone marrow and the positive control after nested PCR (lanes 2 and 3). We confirmed that the amplified products were fragments of drad-1 since the products were cut into two fragments by Msc I (lanes 5 and 6). No product was detected in negative controls (PCR without template cDNA; lanes 4 and 7). Lane 1, fragments of Hinc II digested øx174 DNA as size markers.
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Discussion
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Many hematopoietic growth factors have been identified. However, these factors are insufficient to reconstruct a hematopoiesis-inductive microenvironment in vitro. Hematopoietic stem cells can be maintained by coculture with established stromal cell lines in vitro, whereas stem cells are difficult to maintain without stromal cells. If as-yet-unknown but essential hematopoiesis-regulating factors are provided by the stromal cells, it may be possible to identify such factors among proteins expressed in the stromal cells. In this study, we hoped to identify novel hematopoiesis-regulating factors that are produced by the stromal cells by differential display, and screened mRNA expressed in the hematopoiesis-supporting stromal cells but not in non-hematopoiesis-supporting cells. Such genes may encode proteins that may be candidates for hematopoiesis-supporting factors. We identified eight mRNAs that appeared to be differentially expressed in PA6 cells and A54 preadipocytes but not in A54 adipocytes, and we named one novel gene, drad-1.
As shown in Figures 2D and 3A, we confirmed that drad-1 was expressed in 10T1/2 parent cells, A54 preadipocytes, ST2 cells, and PA6 cells, but the level of expression was low in A54 adipocytes. The bands in the A54 adipocyte fractions ( Fig. 2D and Fig. 3A, lane 3) might have been derived from residual preadipocytes that remained after the induction of terminal differentiation of most cells into adipocytes. In fact, some cells without fat droplets were visible after treatment with insulin and dexamethasone, suggesting that a few preadipocytes remained in the adipocyte fraction. Thus, it is conceivable that drad-1 is not actually expressed by A54 adipocytes. This observation suggests that drad-1 may be a useful molecular marker of adipocyte differentiation.
As shown in Figure 3C, expression of drad-1 was not detected in lymphocytes or myeloid cell lines that we examined. We also examined expression of drad-1 in other tissues, namely heart, brain, lung, liver, skeletal muscle, kidney, and testis, by Northern analysis. Very weak expression of drad-1 was found in lung and kidney, whereas no expression was detected in other tissues (data not shown). As shown in Figure 4, drad-1 was expressed in bone marrow cells. These results suggest that expression of drad-1 is restricted to specific tissues or cell lines.
It is interesting that the pattern of expression of drad-1 resembled that of the hematopoiesis-supporting phenotype in stromal cell lines. A54 preadipocytes, PA6 cell line, and ST2 cell line that have hematopoiesis-supporting ability expressed the drad-1 gene, whereas A54 adipocytes that lost the ability did not express the gene. However, at present, it is not possible to discuss the correlation between hematopoiesis-supporting ability and the expression of drad-1 since we have only limited data.
To further characterize the gene, we are currently screening a library to isolate the entire drad-1 cDNA, and the deduced amino acid sequence of drad-1 should provide some information about the function of the protein.
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Conclusion
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To identify novel hematopoiesis-regulating factors, this study was designed to isolate unknown genes by comparing genes expressed in cell lines with different hematopoiesis-supporting abilities, and we isolated seven novel genes. Among these, one gene was expressed in bone marrow and preadipocytes that have hematopoiesis-supporting ability.
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Acknowledgments
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We thank Miss Y. Mogi for her excellent technical assistance.
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References
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accepted for publication on June 4, 1997.
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Abstract
Introduction
