[Introduction]

Mucins are a group of glycoproteins characterized by tandem repeat domains consisting of high proportion of serines and threonines, through which O-glycosylation occurs. More than 20 mucin genes have been identified. They are largely classified by two groups; secreted mucins and transmembrane mucins. Mucins have been known to be involved in various biological processes. Under normal conditions, they are expressed on the epithelial cell surfaces of respiratory, gastrointestinal or reproductive tracts and function as a protective barrier. Some malignant cells express mucins aberrantly or with abnormal glycosylation patterns, which possibly causes several cellular behavioral changes including altered responses to external signals or modified interactions with other cells. Particularly, several transmembrane mucins were proved to be involved in pathogenesis such as cancers, and further their usefulness as diagnostic or prognostic markers, or therapeutic targets was presented.

As one of transmembrane mucins, Muc21 was recently identified as a molecular entity of epiglycanin, which has long been known for its unique roles on TA3-Ha mouse mammary adenocarcinoma cells. Having a molecular weight of 500 KDa and a length of 500 nm from cell surfaces, epiglycanin has been hypothesized to enable TA3-Ha cells to grow immunologically incompatible hosts and to provide anti-adhesive properties and enhanced malignancies to the cells. However, definite evidence was not obtained because the gene was not identified. In the present work, we show for the first time the functions of Muc21 in cells by overexpressing the identified Muc21 cDNA. Expression.of Muc21 caused striking morphologic changes of the cells through its anti-adhesive property. The underlining mechanism was studied using transient transfection system in HEK293T human embryonic kidney cells. Moreover, the effect of Muc21 expression on cellular behaviors of tumor cells was determined using B16-F1 cells.

[Results and Discussion]

1. Characterization of anti-adhesive property of Muc21

Expression of Muc21 in cells using an artificially constructed cDNA and morphologic changes of the cells

Under the circumstance that Muc21 cDNA was not fully cloned yet, an artificial cDNA was constructed by connecting three fragments; the N-terminal signal sequence, the tandem repeat domain consisting of 84 tandem repeats of 15 amino acids, and the C-terminal cytoplasmic domain. The artificial cDNA was further incorporated into an IRES-Venus vector. When the cDNA was transiently transfected into 293T cells, Muc21 was expressed at the cell surfaces as detected by Muc21 mAb 1A4-1 along with Venus expression (Fig. 1A). By western blotting and lectin blotting, Muc21 was shown to be a large highly glycosylated molecules (Data not shown). Markedly, Venus-positive cells lost their cellular extensions and become round and more than half of the Venus-positive cells became floating (Fig. 1B). In contrast, 293T cells transiently expressing human MUC1 did not show similar morphologic changes indicating that the effect is unique for Muc21. The floating cells were TUNEL negative indicating that the floating phenotypes were not due to undergoing apoptosis (data not shown).

Examination of molecular domains of Muc21 responsible for the non-adherent phenotypes

The structural basis of anti-adhesive properties of Muc21 was investigated. In its cytoplasmic tail, Muc21 has several functional motifs, which might be involved in cellular signaling events leading to the morphologic changes. Thus, cytoplasmic domain-deficient mutants were constructed with an N-terminal flag tag and transfected into 293T cells. The floating phenotype was maintained, although the proportion of the floating cells was reduced compared to Muc21 transfectants having the intact cytoplasmic tail (Fig. 2A). Since the cytoplasmic domain of Muc21 seemed not to be fully responsible for the anti-adhesiveness, the contribution of the extracellular domain of Muc21 was determined. When various Muc21 mutants having different numbers of tandem repeats or no tandem repeats were constructed with an N-terminal Flag-tag and transiently transfected into 293T cells, the percentages of floating cells among the Flag-positive cells closely correlated to the number of tandem repeats (Fig. 2B). Therefore, the large highly glycosylated tandem repeat domain of Muc21 is crucial for inducing the anti-adhesiveness.

Investigation of the mechanism of the anti-adhesive effect induced by Muc21

The mechanism by which the large tandem repeat domain of Muc21 induces anti-adhesive property was explored. To see whether Muc21 interferes cell-cell adhesion or cell-extracellular matrix (ECM) adhesion, over 90% Venus positive cells were collected by cell sorting after transient transfection of the Muc21-IRES-Venus vectors and subjected to homotypic aggregation assay and adhesion assay. Muc21 transfectants having 84 tandem repeats completely prevented cell-cell adhesion (Fig. 3A) as well as cell-ECM components adhesion (Fig. 3B). Muc21 transfectants having 4 tandem repeats partially inhibited homotypic cell adhesion and showed no inhibitory effect on cell-ECM interaction. Because the large tandem repeat domain of Muc21 prevented integrin-dependent cell-ECM interaction, but not integrin-independent cell to poly-lysine interaction , cell surface accessibility of integrin was assessed by the binding of antibody specific for 131 chain of integrin. The antibody binding was significantly reduced by the expression of Muc21 (Fig. 3C), without affecting the protein levels of 81 integrin in whole cell lysates (data not shown) indicating that interference of integrin function might play an important role in the anti-adhesive effect. Sialic acids on the tandem repeats of Muc21 provide negative charges which might induce anti-adhesive effect by charge repulsion. The contribution of sialic acids was determined by performing homotypic aggregation assay and adhesion assay after the treatment of sialidase . Sialidase treated cells and untreated cells showed similar anti-adhesive property (Fig. 3A and 3B) showing negligible contribution of sialic acids. Collectively, these results indicate that Muc21 induce anti-adhesive effect through its large tandem repeat domain possibly by preventing the interaction of cell adhesion-related molecules with its counterpart.

2. Examination of tumor cell behavioral changes induced by Muc21 expression in vitro and in vivo

In vitro cellular behavioral changes induced by Muc21 expression

To determine how Muc21 contributes to malignant cell behaviors, B16-F1 melanoma cells were transfected with the Muc21-IRES-Venus vectors and stable transfectants were obtained by Venus positive-cell sorting and cloning. The clones with high expression of Muc21 showed higher percentages of floating cells similar to the 293T/Muc21 transfectants. To examine whether Muc21 expression provide survival or growth advantages in these cells, poly(2-hydroxyethylmethacrylate) (poly-Hema) coated plates were used as non-adherent surfaces. B16-F1/Muc21-84TR transfectants showed increased cell growth compared to B16-F1/mock transfectants (Fig. 4A). In the B16-F1/mock cells, cell viability was reduced as incubation time increased on the poly-Hema coated plates whereas the viability of B16-F1/Muc21 transfectants was not affected even after prolonged incubation under the same condition (Fig.4B). These results suggest that Muc21 expression not only rendered cells to be non-adherent but also contributed to enhanced survival under non-adherent conditions.

The masking role of Muc21 on the tumor cell surfaces could provide escape mechanisms by inhibiting tumor cell interactions with cells in the immune system. To prove or disprove this hypothesis, cytotoxic effects by IL-2 activated splenocytes on B16-F1/Muc21 transfectants were examined. B16-F1/Muc21 transfectants showed substantially reduced sensitivity to cytotoxic effects than mock transfectants. The reduction was apparently dependent on the size of tandem repeats (Fig. 4C). Strong anti-adhesive property of Muc21 might prevent tumor cells from interaction with endothelial cells. Using SvBCE bovine corneal endothelial cells, B16-F1/Muc21 transfectants showed notably reduced adhesion to the endothelial cells (Fig. 4D).

In vivo cellular behavioral changes induced by Muc21 exaression

Two well known in vivo experimental models for B16-F1 melanoma cells were applied to examine whether Muc21 expression could change tumor cell behaviors in vivo. In intravenous lung metastasis model which has been known to strongly reflect adhesive or invasive properties of tumor cells, Muc21 expression showed a negative effect on metastasis formation. Three weeks after i.v. injection of transfectants, the numbers of lung metastases from B16-F1/Muc21 transfectants were smaller than those from mock transfectants (Fig. 5A). These results suggest that reduced tumor cell adhesion to endothelial cells by Muc21 expression probably led to diminished metastasis formation. In subcutaneous model, Muc21 expression did not affect tumor growth (Fig. 5B) indicating a possible correlation with in vitro results of a comparable anchorage-dependent growth between mock and Muc21 transfectants (Data not shown).

3. Production of Muc21 gene deficient mice and verification of their Muc21 gene expression (Data not shown)

In a means to investigate the function of Muc21 under normal and .pathological states, the construction of Muc21 gene deficient mice was attempted. Exon I including the transcription initiation region was targeted to be replaced by a neomycin gene and the correctly targeted ES cells were used for the production of chimeric mice. The disruption of exon I was confirmed by PCR or RT PCR in homozygous Muc21 gene deficient mice. Unexpectedly, Muc21 transcripts were identified in C-terminal region of exon II and exon III. Using 5'-RACE and subsequent CR, transcripts encoding more than 15 tandem repeats close to the C-terminal region were recognized. By immunohistochemistry, mAb 1A4-1 stained esophagi and vagina from the Muc21 gene deficient mice after sialidase treatment. Considering the fact that the specificity of mAb 1A4-1 to the Muc21 protein is not clearly defined yet, extinction or existence of the Muc21 protein in the Muc21 gene deficient mice should be further elucidated.

[Conclusion]

Muc21 expression substantially changed cellular behaviors in vitro and in vivo. The observed strong anti-adhesive property of Muc21 suggest that Muc21 expression might affect various biological processes including prevention of epithelial cells from interaction with pathogens or detachment of cancer cells from surrounding cells or ECM to initiate metastasis. The in vitro results using B16-F1/Muc21 transfectant cells further suggest that Muc21 might be involved in enhanced tumor cells survival. Further research to correlate the in vitro behaviors of Muc21 transfectants with in vivo behaviors should be done. This is the first report on the functions of a novel transmembrane mucin Muc21. We believe this work will provide the basis for the further research on the functions of Muc21 in immunology and cancer biology.

Fig. 1. Non-adhesive phenotypes induced by Muc21 expression. The Muc21-IRES-Venus vector was transfected into 293T cells and Muc21 expression was detected with mAb1A4-1 (A). The floating cells were collected from culture media and counted (B).

Fig. 2. Tandem repeat dependency of non-adhesive phenotypes. Cytoplasmic domain deficient mutants were constructed with an N-terminal Flag-tag and transfected into 293T cells (A). Extracellular domain mutants were constructed and transfected into 293T cells (B). Note that the mutants in B were constructed using a different vector system from mutants in A.

Fig. 3. Masking of cell surface adhesion-related molecules by Muc21. Venus positive cells were allowed to aggregate by rotating at 37 degree and aggregates formation was evaluated under microscope (A). Venus positive cells were allowed to adhere to ECM components for 3 hr and adherent cells were measured by crystal violet assay (B). Transient transfectants were assayed by flow cytometry using integrin B1 antibody (C). Sial+; sialidase treated cells, Sial-; sialidase untreated cells

Fig. 4. In vitro tumor cells behavioral changes induced by Muc21. Stable transfectant clones in B16-F1 cells were seeded on the poly-Hema coated plates and cell growth was measured by a cell counting kit, CCK-8 (A). Viability of transfectants grown on the poly-Hema coated plates was measured by trypan blue exclusion assay (B). Ligand-labeled transfectants were incubated with IL-2 activated splenocytes for 2 hr and ligand release was detected by fluorescence (C). Transfectants were allowed to adhere to endothelial cells for 40 min and adherent cells were evaluated with flow cytometry by detecting Venus-positive cells (D).

Fig. 5. In vivo tumor cell behavioral changes induced by Muc21. Transfectants were injected into tail vein of syngeneic mice and lung metastatic foci were counted after 3 weeks (A) (n=10). Transfectants were injected into the right flanks of syngeneic mice and tumor size were measured ever other day (B) (n=5).

「Biological characterization of cells expressing Muc21/epiglycanin(Muc21/epiglycanin発現細胞の生物学的な特性)」と題する本論文は、癌細胞表面ムチンとして1975年に記載されたエピグリカニンを対象に、これを発現する細胞の挙動を明らかにした結果を述べたものである。エピグリカニンはマウス乳がん細胞株であるTA3-Haの表面に見出され、別の亜株で造腫瘍性・可移植性の低いTA3-Stには発現していなかったことから、癌細胞の挙動の違いをもたらす原因分子の一つと考えられて来た。さらに、TA3-Ha細胞は宿主の免疫応答に基づく癌細胞排除機構に抵抗性を示し、これもエピグリカニンが細胞表面を被覆するためであると仮定されていた。これらの報告はその後発見されたMUC1をはじめとする腫瘍細胞表面ムチンを発現する癌が予後不良である原因を考察する上で、しばしば引用されて来た。しかし、エピグリカニンの遺伝子が同定されていなかったため確固たる証明ができないまま30年余が経過していた。エピグリカニンの遺伝子が同定されMuc21として確認されたが、強制発現細胞を作製してその機能を明らかにするにはいくつかの障壁があった。先ず、この巨大な分子の全長ORFは取得されていなかった。また、発現したタンパク質には約800の糖付加部位があり、最終的に細胞表面に存在する分子は重量比として70%以上が糖である可能性が高いが、この部分の構造多型(グリコフォーム)を簡便に分別検出する方法が当初無かった。学位申請者は,第一の問題を、新たに全長遺伝子を,それぞれクローニングした3部分から人工的に再構築して発現ベクターを作製して解決し、第二の問題をグリコフォーム特異的なモノクローナル抗体を開発することで、部分的にではあるが解決し、発現細胞の挙動解析を可能にした。対象としては、ヒト胎生腎細胞由来HEK293T細胞とマウスメラノーマB16-Fl細胞を用い、これらにMuc21を一過的または安定に発現させて、細胞の性質の変化を調べた。

HEK293T細胞にVenusベクターを用いてMuc21を一過性に発現させ、蛍光を発する細胞を観察した。その結果、Muc21発現によって細胞が接着状態から浮遊状態に変化することを明らかにした。この変化はMuc21の細胞質ドメインを完全に欠失させても起こるが、糖付加を受ける配列の繰り返しであるタンデムリピートドメインを欠失させると欠失部分の割合に比例して細胞を浮遊化する能力が失われたので細胞外の糖鎖を含む部分が重要であることが明らかとなった。Muc21を強制発現させた細胞では同じ細胞どうしの接着性や、フィプロネクチン、ラミニン、IV 型コラーゲン、及び基底膜のモデルであるマトリゲルへの接着性が低下しており、このような低下はタンデムリピートドメインを短小化したMuc21ではわずかしか見られなかった(84回繰り返しと4回繰り返しを含むものの比較に基づく)。しかし、ポリーLーリジンへの接着性には影響がなかったのでMuc21の影響は、特定の接着分子の機能を修飾していると考えられた。実際に、抗インテグリンβ1鎖抗体のMuc21を強制発現させた細胞への結合性を見ると有意に低下しており、タンデムリピート部分を欠損させたMuc21にはこの効果は見られなかった。また、細胞を可溶化し、ウエスタンブロッティング法でインテグリンβ1鎖を検出すると、Muc21強制発現細胞において、非発現細胞と同程度の量が検出された。Muc21によるインテグリンの機能低下または細胞表面におけるアクセシビリティーの低下が細胞浮遊化の原因であることが明らかとなった。293T細胞に発現したMuc21は主にガラクトースを末端に持つ短小な糖鎖であるが、シアル酸を持つ糖鎖も含まれることがモノクローナル抗体やレクチンによる解析で明らかになった。そこで抗接着効果が陰性荷電を持つシアル酸に依存する可能性を調べるため、細胞をシアリダーゼ処理した後に接着実験を行った。その結果糖鎖末端のシアル酸残基はMuc21による細胞浮遊化に貢献していないことが確かめられた。

細胞の増殖や生存へのMuc21の影響は、B16-F1メラノーマ細胞を背景とするMuc21安定発現細胞クローンを複数作製し、比較することによって検証した。ここでもタンデムリピート84回繰り返しと4回繰り返しを比較した。これらの細胞の足場非依存的増殖性を比較するためにポリヒドロキシエチルメタクリル酸(poly-HEMA)処理した表面上で培養を行った。その結果タンデムリピート84回の繰り返しを持つMuc21強制発現細胞では足場非依存的な増殖性が獲得されていることが示された。さらに、Muc21発現のない細胞では足場非依存状態で経時的にアポトーシスが誘導されるが、強制発現細胞では足場のない状態で高い生存率を保つことが明らかになった。Muc21発現B16-F1メラノーマ細胞は、接着性の低下に起因する細胞相互作用の変化が見られると考えられたが、実際にナチュラルキラー細胞による殺傷効果に対して抵抗性が高い、血管内皮細胞に対する接着性が低い、などの非発現細胞とは異なる性質が認められた。これらの細胞のin vivoにおける振る舞いを、尾静注後の肺転移形成能と皮下注後の造腫瘍能に関して比較した。前者の能力はMuc21発現細胞では低下しており、後者の能力は変化していなかった。以上より、タンデムリピート84回繰り返しのムチン領域を含むMuc21を強制発現させたB16-F1細胞は、少なくともin vitroでは生存が脅かされる条件下で生存しやすくなっていることが判明した。一方in vivoでは、悪性挙動が増強しているわけではなかった。

これらの結果は、腫瘍ムチンの細胞表面における機能について、徹底的にまたその構造的特徴に注意を払って解析したものである。それらの研究成果は新規ムチンMuc21の機能を明らかにしたばかりでなく、癌細胞の表面分子として量的に豊富で特徴的な構造を持つムチンが、癌細胞の振る舞いにどのような影響を与えるかに関して新しい知見を与えるものであり、糖鎖生物学、腫瘍学、及び免疫学に資するところが大である。よって、これらの研究を行った李ユリは博士(薬学)の学位を得るにふさわしいと判断した。