CD43, also known as leukosialin and sialophorin, is a type-Ia transmembrane molecule critical to leukocyte activation and adhesion.1-3 The N-terminal extracellular region is a rod-like structure of 235 amino acids that is heavily glycosylated and extends 45nm from the cell surface. The transmembrane region comprises 23 amino acids, and the intracellular region of 123 amino acids anchors the protein to the cytoskeleton by binding actin, ezrin, and moesin.
CD43 has been described as a Janus molecule after the Roman god with two faces.2 This analogy reflects the finding that CD43 can perform diametrically opposite functions. First, depending upon how it is engaged at the cell surface, CD43 can either induce or protect against leukocyte apoptosis. Second, depending upon the status of leukocyte activation, CD43 can act either as an anti-adhesion barrier molecule or a pro-adhesion receptor.
While leukocytes are at rest, the length, bulk, and strong negative charge of the extracellular domain of CD43 combine to inhibit adhesion and maintain leukocytes in the circulation. During leukocyte activation, expression of CD43 is reduced by proteolysis and repression of its gene. In addition, CD43 is excluded from foci of cell-cell contact. Together, this down-regulation and redistribution of CD43 facilitate intercellular interaction effected by other leukocyte molecules such as the β2 integrins. Furthermore, changes in glycosylation of the extracellular region allow the CD43 that remains at the cell surface to act as a pro-adhesive counter receptor for galectin-1, ICAM-1, E-selectin, sialoadhesin, and MHC Class I.
During leukocyte activation, cleavage of the extracellular domain triggers subsequent cleavage of the intracellular domain and its translocation to the nucleus, where it protects against apoptosis and drives proliferation by binding β-catenin. In addition, the intracellular domain mediates activation signals by binding tyrosine kinases Fyn and Lck and the serine/threonine kinase STANK. Activation signals transduced by CD43 can lead to phosphorylation of Shc, Syk, Lyn, Vav, PLCγ2, SLP-76, CD3ζ, PKM2, Bad, and STAT3. These events result in activation of Lck, Fyn, Src, PI3K, p38, MAPK, and PKC, repression of the Ras inhibitor Cbl, and translocation of ERK2 and ERK5 into the nucleus. Ultimately, the intracellular signaling cascades mediated by CD43 induce DNA-binding of AP-1, CREB, NFAT, and NF-kB and increased expression of IL2, IL4, IL4R, IFNγ, IFNγR, CD69, CD40-L, c-Myc, and cyclinD1.
Historically, CD43 expression was reported to be restricted to leukocytes and platelets.1-3 However, we and others have shown that CD43 is also expressed in lung, cervix, breast, colon, and salivary gland cancers, where it drives malignant progression.4-14 During the course of an immunohistochemical screen of additional cancers, we found that malignant testicular tissue also expressed CD43. However, unexpectedly, we also found CD43 expression by apparently normal adjacent tissue. Cancer can change the gene expression profile of nearby normal tissue. Consequently, we analyzed entirely non-malignant testis tissue that had been resected from patients who had suffered accidental trauma. This analysis demonstrated that the N-terminus of CD43 is expressed in the cytoplasm of normal Sertoli and Leydig cells, while the C-terminus is expressed in the nucleus of normal Sertoli cells, spermatogonia, and pachytene spermatocytes, but is absent from spermatids. Therefore, as germ cells differentiate, CD43 appears to be progressively down-regulated. Also, since the patterns of N and C-terminal expression do not overlap, these domains seem to exist in testes as separate entities analogous to what occurs during normal leukocyte activation and breast cancer. Further analysis indicates that testicular CD43 is found in spherical cytoplasmic and punctate nuclear structures.
In order to begin to determine the function of CD43 in the testis, we generated two daughter lines from the human germ cell line TCam-2. The first daughter, named TCam-43(+), stably expresses a scrambled ineffective shRNA, and the second, named TCam-43(-), stably expresses four shRNAs complementary to different regions of CD43 mRNA (OriGene Technologies, Inc., Rockville, MD). Western blotting demonstrated that CD43 expression was effectively knocked down in TCam-43(-) compared to TCam-43(+). This down-regulation mimics CD43 repression observed during germ cell differentiation in vivo. CD43 repression appears to be a driver of germ cell differentiation as it causes an increase in the molecular weight of transition protein 1 (TNP1) and changes in acrosin expression. The increase in TNP1 molecular weight is consistent with CD43 repression inducing post-translational modifications such as serine phosphorylation, arginine or lysine methylation, and/or lysine acetylation. These modifications target TNP1 for complete replacement with protamines that allow formation of highly condensed chromatin in mature sperm. Changes in acrosin expression include a shift to a lower molecular weight form consistent with the processing of preproacrosin to proacrosin and a shift from cytoplasmic to nuclear expression consistent with translocation from the Golgi apparatus to the acrosome cap of the mature sperm nucleus.
Next, we sought to determine if testicular CD43 expression is evolutionarily conserved. This was demonstrated by immunohistochemical analysis of the mouse Leydig cell line TM3. Mimicking human Leydig cells in vivo, the N-terminal domain of TM3 CD43 is expressed in the cytoplasm and in vesicle-like structures.
In order to begin to determine the function of CD43 in mouse Leydig cells, we generated two daughter lines from the mouse Leydig cell line MLTC-1. The first daughter, named MLTC-43(+), stably expresses a scrambled ineffective shRNA, and the second, named MLTC-43(-), stably expresses four shRNAs complementary to different regions of mature CD43 mRNA (OriGene Technologies, Inc., Rockville, MD). When the culture media conditioned by these daughter lines was analyzed, CD43 repression was found to induce estradiol, testosterone, and progesterone secretion by 32%, 60%, and 53%, respectively (P < 0.01).
DISCUSSION
Immunohistochemical analysis of the human testis demonstrates that the N-terminal domain of CD43 is expressed in the cytoplasm of Sertoli and Leydig cells, while the C-terminal domain is expressed separately in the nucleus of Sertoli and germ cells. The mechanisms by which these CD43 moieties are generated are unknown. At the RNA level, possible mechanisms include the use of alternative transcription initiation sites, various forms of RNA editing, differential splicing, and/or the utilization of alternative polyadenylation signals. At the protein level, differential expression of the N and C terminals of CD43 could be caused by the use of alternative translation initiation and termination signals and/or proteolytic cleavage events. In addition, like leukocyte CD43, the testicular function of CD43 could be controlled by differential phosphorylation, glycosylation, sumoylation, and/or subcellular localization.
The N-terminal domain of CD43 is expressed in punctate structures within the cytoplasm of Sertoli and Leydig cells, while the C-terminal domain is expressed in punctate structures within the nucleus of Sertoli and germ cells. In leukocytes, CD43 can localize to exosomes, endosomes, microvesicles, vacuoles, and PML nuclear bodies. Furthermore, breast cancer cells secrete CD43 in exosomes. Similar entities likely represent the punctate structures that harbor CD43 in testicular cells. The functional role played by CD43 in these structures remains to be determined.
The function of Leydig cells is controlled in large part by luteinizing hormone (LH), an intrinsic circadian clock, and crosstalk secretions with Sertoli and germ cells. Major control of Sertoli cell function is exerted by follicle-stimulating hormone (FSH) and crosstalk secretions with Leydig and germ cells. Germ cell differentiation is dependent upon crosstalk secretions with Leydig and Sertoli cells and can be mimicked in vitro by the combined action of epidermal growth factor (EGF), fibroblast growth factor-4 (FGF-4), transforming growth factor-β (TGF-β,), and bone morphogenetic protein-4 (BMP-4).
With regard to the endocrine, paracrine, and circadian control of testicular CD43 gene expression, it is important to note that the nucleotide sequences of the promoter regions of the human and mouse CD43 genes are evolutionarily conserved and both contain elements that could confer circadian transcription by binding CLOCK:BMAL1 and members of the REV-ERB and ROR nuclear receptor families. In addition, it is intriguing that in both the mouse and human genome the CD43 gene overlaps or is closely linked with the genes encoding epididymis secretory sperm binding protein Li 90n and zymogen granule protein 16 implicated in sperm capacitation.
CD43 expression represents new biology for the human testes with potential implications for the development of new approaches to the treatment of male infertility. In about one-third of cases, the etiology of male infertility remains unknown and is termed idiopathic male infertility. Despite the fact that these men have no history of diseases affecting fertility and show normal findings in physical examinations and endocrine, genetic, and biochemical laboratory testing, their semen analysis often reveals abnormal semen parameters. In addition, patients with idiopathic male infertility often exhibit germ cell aplasia or Sertoli cell-only syndrome (SCOS). This condition is characterized by severely reduced or absent spermatogenesis despite the presence of both Sertoli and Leydig cells. It will be of interest to determine if idiopathic male infertility is characterized by abnormal testicular expression of CD43. If this proves to be the case, correction of such abnormal expression would represent a novel therapeutic strategy. Conversely, drugs causing a pattern of CD43 expression mimicking that in idiopathic male infertility would represent a novel means of male contraception.
Semen analysis is the test of choice for assessing the male partner in an infertile couple. However, a major limitation of semen analysis is its emphasis on sperm evaluation over the characterization of seminal plasma, which represents 95% of the ejaculate. Seminal plasma is constituted by secretions derived from the testes, epididymis, and seminal vesicles, as well as the prostate, bulbouretheral and periurethral glands. Analysis of human seminal plasma has begun to yield biomarkers for male fertility and infertility. Most proteins in seminal plasma bind to the sperm surface through exosomes, where they modulate sperm function, interaction with the female reproductive tract, and, finally, fertilization. CD43 has been found in exosomes secreted by breast cancer endothelium. In addition, the N-terminal domain of leukocyte CD43 can be shed into blood plasma, where it is known as galactoglycoprotein. These findings indicate that CD43 moieties likely form constituents of seminal plasma, and alterations in their expression could represent noninvasive biomarkers of male infertility. In this regard, it is also important to note that male mice in which the CD43 gene has been knocked out are fertile. Consequently, male infertility is likely to be characterized by the abnormal presence, not the absence, of CD43.
CONCLUSIONS
We report for the first time that CD43 is expressed in normal human testes. During germ cell differentiation, CD43 is down-regulated, driving changes in the expression of both transition protein 1 and acrosin consistent with spermatid maturation. In addition, down-regulation of CD43 in Leydig cells induces secretion of steroid hormones. The exact molecular mechanisms by which CD43 affects these changes in germ and Leydig cells remain to be determined. Nevertheless, based on our current data, we tender the hypotheses that CD43 actively inhibits testicular function, its aberrant expression may contribute to male infertility, and manipulation of its expression may represent a new means of treatment and contraception. In addition, specific patterns of CD43 expression in the seminal plasma could predict the success of conventional in vitro fertilization or intracytoplasmic sperm injection. Embryo transfer during assisted reproduction is significantly improved by exposure to seminal plasma. Consequently, targeting molecular defects in seminal plasma CD43 might have therapeutic benefit.
Written by: Carl Simon Shelley,1 & Qiangwei Fu,2
- Leukemia Therapeutics, LLC, Hull, MA, USA.
- California Institute for Biomedical Research, La Jolla, CA, USA.
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