CD9-null Mice Research Articles

A role for tetraspanin proteins in regulating fusion induced by Burkholderia thailandensis

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbβ3 expression and clot retraction in platelets

Integrin αIIbβ3 is critical for platelet-mediated blood clotting. Tetraspanins are well-established regulators of integrins and genetic loss of tetraspanin CD151 or TSSC6 in mice leads to increased bleeding due to inadequate integrin αIIbβ3 outside-in signaling. Conversely, mild but enhanced integrin αIIbβ3 activation and hyperaggregation is observed in CD9 and CD63 null mice respectively. CD82 is reportedly expressed in platelets; however its function is unknown. Using genetically engineered CD82 null mice, we investigated the role of the tetraspanin CD82 in platelet activation. Loss of CD82 resulted in reduced bleed times in vivo. CD82 was present on the surface of both human and mouse platelets, and its levels did not change upon platelet activation or degranulation. No differences in platelet activation, degranulation, or aggregation in response to ADP or collagen were detected in CD82 null mice. However, the kinetics of clot retraction was enhanced, which was intrinsic to the CD82-null platelets. Integrin αIIbβ3 surface expression was elevated on the platelets from CD82 null mice and they displayed enhanced adhesion and tyrosine kinase signaling on fibrinogen. This is the first report on CD82 function in platelets; which we found intrinsically modulates clot retraction, integrin αIIbβ3 expression, cell adhesion, and tyrosine signaling.

Downregulation of CD9 in Keratinocyte Contributes to Cell Migration via Upregulation of Matrix Metalloproteinase-9

Tetraspanin CD9 has been implicated in various cellular and physiological processes, including cell migration. In our previous study, we found that wound repair is delayed in CD9-null mice, suggesting that CD9 is critical for cutaneous wound healing. However, many cell types, including immune cells, endothelial cells, keratinocytes and fibroblasts undergo marked changes in gene expression and phenotype, leading to cell proliferation, migration and differentiation during wound repair, whether CD9 regulates kerationcytes migration directly remains unclear. In this study, we showed that the expression of CD9 was downregulated in migrating keratinocytes during wound repair in vivo and in vitro. Recombinant adenovirus vector for CD9 silencing or overexpressing was constructed and used to infect HaCaT cells. Using cell scratch wound assay and cell migration assay, we have also demonstrated that downregulation of CD9 promoted keratinocyte migration in vitro, whereas CD9 overexpression inhibited cell migration. Moreover, CD9 inversely regulated the activity and expression of MMP-9 in keratinocytes, which was involved in CD9-regulated keratinocyte migration. Importantly, CD9 silencing-activated JNK signaling was accompanied by the upregulation of MMP-9 activity and expression. Coincidentally, we found that SP600125, a JNK pathway inhibitor, decreased the activity and expression of MMP-9 of CD9-silenced HaCaT cells. Thus, our results suggest that CD9 is downregulated in migrating keratinocytes in vivo and in vitro, and a low level of CD9 promotes keratinocyte migration in vitro, in which the regulation of MMP-9 through the JNK pathway plays an important role.

CD9 Is Critical for Cutaneous Wound Healing through JNK Signaling

Cutaneous injury triggers a cascade of signaling events essential for wound re-epithelialization. CD9, a cell-surface protein, has been implicated in a number of cellular processes by coupling to intracellular signaling; however, its exact role in wound healing remains unidentified. We reported that CD9 was downregulated in migrating epidermis, and reelevated to basal level when re-epithelialization was completed. Although low level of CD9 appears to be required for normal wound healing, a significant healing delay was found in CD9-null mice, with wounds gaping wider on day 5 and day 7 post wounding. Further analysis showed that re-epithelialization was adversely affected in CD9-null mice, due to impaired migration of epidermis. Notably, CD9 deficiency caused a persistent enhancement of C-JUN NH2 terminal kinase (JNK) signaling primarily in migrating epidermis with abnormal elevation of matrix metalloproteinase (MMP)-9 detected in CD9-null wounds, leading to excessive degradation of type IV collagen, and thus a defective basement membrane at the wound site. JNK suppression reduced MMP-9 production and therefore ameliorated the healing delay with the appearance of significantly elongated migrating epidermis in CD9-null mice. Our study demonstrated the importance of CD9 in wound re-epithelialization, linking this molecule directly to basement membrane formation and epidermal migration through participating in the regulation of the JNK/MMP-9 pathway.

The Critical Function of c-Myb and CD9 On Megakaryopoiesis.

Abstract 4587The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We previously reported that the c-Myb knock down (KD) mice exhibited anemia and thrombocytosis. To better understand the transcriptional regulatory program that accompanies the decrease of c-Myb, we performed a transcriptome analysis of megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). We found that CD9 expression was dramatically increased in the MEPs from c-Myb KD mice. CD9 belongs to a tetraspanin family and regulates cell motility and adhesion. Several reports described that CD9 is involved in platelet function by forming a complex with integrin family members including platelet fibrinogen receptor integrin aIIb-b III. To elucidate the functional contribution of CD9 to the thrombogenesis within the regulatory hierarchy mediated by c-Myb, we first examine the relation between c-Myb and CD9. We found the recruitment of c-Myb to the promoter region of CD9 by chromatin immunoprecipitation assay. A reporter assay showed that c-Myb represses CD9 promoter activity. These results indicate that CD9 is directly repressed by c-Myb. Since an agonistic antibody against CD9 stimulated megakaryocytic colony formation, we investigated the role of CD9 during megakaryopoiesis in vivo by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Consistent with this result, the recovery phase of platelet counts were delayed in the CD9-null mice after the bone marrow suppression with 5-fluorouracil, indicating the impaired platelet production. In CD9-null mice, the proliferation of megakaryocytes was promoted and circulating megakaryocytes in the peripheral blood were increased, which might compensate the PPF impairment of CD9-null megakaryocytes. Our study suggests that c-Myb suppresses CD9 expression under the steady state, while, in the stress megakaryopoiesis, CD9 is derepressed and acts to enhance platelet production. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis. Disclosures:Ninomiya:Alexion: Research Funding.

Loss of surface EWI‐2 on CD9 null oocytes

CD9, a member of the tetraspanin family, associates with a variety of other proteins to form the tetraspanin web. CD9 forms direct and relatively stable associations with the immunoglobulin superfamily proteins EWI-2 and EWI-F. Deletion of the Cd9 gene results in female infertility since Cd9 null mice produce oocytes that fail to fuse. It is thought that the absence of CD9 causes the inability of the oocytes to fuse. In this study, we report that the expression level of EWI-2 on the Cd9(-/-) oocyte surface is <10% of the wild-type level. Hence, the severe reduction in EWI-2 activity may be responsible for the loss of fusion ability. An entirely different mutant of CD9, not a deletion but a depalmitoylated construct, does not affect in vivo female fertility suggesting that the palmitate modification of CD9 is not essential for its putative fusion function. Additionally, the level of EWI-2 on the surface of the oocytes from these females was comparable to the EWI-2 level on wild-type oocytes. We also found that soluble, recombinant EWI-2 binds preferentially to acrosome-reacted sperm but the bound EWI-2 does not inhibit sperm-oocyte fusion. Overall, the results indicate that deletion of CD9, which is known to have multiple associations, may have pleiotropic effects on function that will require further dissection.

Functional Analysis of CD9 during Megakaryopoiesis

CD9 is a four transmembrane protein belonging to a tetraspanin family and regulates cell motility and adhesion. Several reports have indicated that CD9 form complexes with integrin including platelet fibrinogen receptor integrin aIIb-b III and involve in platelet function. We have previously reported that the c-myb knock down (KD) mice exhibited anemia and thrombocytopenia, and the expression level of CD9 mRNA was markdly increased in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified and agonistic antibody of CD9 stimulated megakaryocytic colony formation. These observations suggested that the CD9 expression was downregulated by c-myb. In our current study, we investigated the role of CD9 during megakaryopoiesis and platelet function by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Furthermore, after the bone marrow suppression with 5-fluorouracil (5-Fu), the recovery phase of platelet counts were delayed in the CD9-null mice. To clarify the reason of this platelet- recover delay, the number of bone marrow megakaryocyte was investigated using anti-vWF antibody staining, but the serial measurement of megakaryocyte number in CD9-null mice was not changed compared with wild-type mice. And also megakaryocyte ploidy was not changed in CD9-null mice compared with wild-type mice. Previous reports revealed that the cytoskeleton reorganization has a key role for the PPF formation, and Rac1-PAK1 signaling is important for actin restructure and aggregation prior to the cytoskeleton reorganization. The PPF formation was suppressed by Rac1 inhibitor. Although the mRNA expression levels of Rac1 in CD9-null mice were almost same as that in wild-type mice, PAK1 activation, major target of Rac1, was delayed in CD9-null mice compared with wild-type mice using thrombin-stimulating platelets. These results suggested that the delay of PAK1 activation cause the suppression of PPF formation in CD9-null mice.Our previous and current studies demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryocytosis, CD9 is upregulated and acts to induce megakaryopoiesis and platelets production through Rac1-PAK1 signaling pathway. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

Delayed wound healing in CD‐9 null mice

Delayed wound healing in CD‐9 null mice

C-Myb Regulates CD9 Gene Expression during Megakaryopoiesis.

The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival, and differentiation of hematopoietic cells. We previously generated through insertion mutagenesis a c-myb gene knockdown (KD) line of mice. In the mice transgene was inserted 77-kb upstream of the c-myb gene and c-Myb expression was markedly decreased in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs) of the homozygous knockdown mutant mice (c-myb KD mice). The c-myb KD mice exhibited anemia, thrombocythemia, and splenomegaly and these abnormalities were reproducible in a co-culture assay of MEPs with OP9 cells, but abrogated by the retroviral expression of c-Myb in MEPs. To understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis with MEPs and identified 74 genes that are upregulated and 36 genes that are downregulated in the c-myb KD mice. Of these genes, expression levels 15 genes are actually changed significantly in bone marrow cells of the c-myb KD mice. These genes harbor c-Myb recognition elements in their regulatory regions. Especially, we found that the CD9 expression was upregulated in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified using a luciferase reporter assay and chromatin immunoprecipitation assay. Agonistic antibody of CD9 stimulated megakaryocytic colony formation. On the contrary, upon the bone marrow suppression with 5-fluorouracil recovery of platelet number was delayed in the CD9-null mice. Furthermore, proplatelet formation was impaired when we used CD9-null mouse megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. These results thus demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryopoiesis CD9 is derepressed and acts to induce the megakaryopoiesis. Elucidation of c-myb-based transcription network seems to be of important to understand the megakaryocytic differentiation.

The CD9 Tetraspanin Is Not Required for the Development of Peripheral B Cells or for Humoral Immunity

The CD9 tetraspanin is known to be expressed at high levels on marginal zone (MZ) B cells, B-1 B cells, and plasma cells, and its expression is believed to be dependent on signals derived via Btk. In CD9 null mice, however, the development and survival of MZ B cells, B-1 B cells, and plasma cells all appear to be unaffected, and humoral immune responses to T-dependent and T-independent Ags are similar to those seen in wild-type littermate controls. In wild-type mice, CD9 levels may serve to distinguish between the presumed MZ precursor B cell population in the spleen and other IgD-expressing transitional B cells that express lower levels of CD21 and CD1d. These results suggest that CD9 is dispensable for B cell development and humoral immunity, but that this protein may serve as an additional marker for the presumed MZ precursor population of splenic B cells.

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.