The tetraspanin CD82 regulates bone marrow homing and engraftment of hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cell (HSPC) transplantation represents a treatment option for patients with malignant and nonmalignant hematological diseases. Initial steps in transplantation involve the bone marrow homing and engraftment of peripheral blood-injected HSPCs. In recent work, we identified the tetraspanin CD82 as a potential regulator of HSPC homing to the bone marrow, although its mechanism remains unclear. In the present study, using a CD82 knockout (CD82KO) mouse model, we determined that CD82 modulates HSPC bone marrow maintenance, homing, and engraftment. Bone marrow characterization identified a significant decrease in the number of long-term hematopoietic stem cells in the CD82KO mice, which we linked to cell cycle activation and reduced stem cell quiescence. Additionally, we demonstrate that CD82 deficiency disrupts bone marrow homing and engraftment, with in vitro analysis identifying further defects in migration and cell spreading. Moreover, we find that the CD82KO HSPC homing defect is due at least in part to the hyperactivation of Rac1, as Rac1 inhibition rescues homing capacity. Together, these data provide evidence that CD82 is an important regulator of HSPC bone marrow maintenance, homing, and engraftment and suggest exploiting the CD82 scaffold as a therapeutic target for improved efficacy of stem cell transplants.

Global deletion of tetraspanin CD82 attenuates bone growth and enhances bone marrow adipogenesis.

CD82 is a widely expressed member of the tetraspanin family of transmembrane proteins known to control cell signaling, adhesion, and migration. Tetraspanin CD82 is induced over 9-fold during osteoclast differentiation in vitro; however, its role in bone homeostasis is unknown. A globally deleted CD82 mouse model was used to assess the bone phenotype. Based on microCT and 4-point bending tests, CD82-deficient bones are smaller in diameter and weaker, but display no changes in bone density. Histomorphometry shows a decrease in size, erosion perimeter, and number of osteoclasts in situ, with a corresponding increase in trabecular surface area, specifically in male mice. Male-specific alterations are observed in trabecular structure by microCT and in vitro differentiated osteoclasts are morphologically abnormal. Histomorphometry did not reveal a significant reduction in osteoblast number; however, dynamic labeling reveals a significant decrease in bone growth. Consistent with defects in OB function, OB differentiation and mineralization are defective in vitro, whereas adipogenesis is enhanced. There is a corresponding increase in bone marrow adipocytes in situ. Thus, combined defects in both osteoclasts and osteoblasts can account for the observed bone phenotypes, and suggests a role for CD82 in both bone mesenchyme and myeloid cells.

CD82/KAI1 Maintains the Dormancy of Long-Term Hematopoietic Stem Cells through Interaction with DARC-Expressing Macrophages.

Hematopoiesis is regulated by crosstalk between long-term repopulating hematopoietic stem cells (LT-HSCs) and supporting niche cells in the bone marrow (BM). Here, we examine the role of CD82/KAI1 in niche-mediated LT-HSC maintenance. We found that CD82/KAI1 is expressed predominantly on LT-HSCs and rarely on other hematopoietic stem-progenitor cells (HSPCs). In Cd82(-/-) mice, LT-HSCs were selectively lost as they exited from quiescence and differentiated. Mechanistically, CD82-based TGF-ß1/Smad3 signaling leads to induction of CDK inhibitors and cell-cycle inhibition. The CD82 binding partner DARC/CD234 is expressed on macrophages and stabilizes CD82 on LT-HSCs, promoting their quiescence. When DARC(+) BM macrophages were ablated, the level of surface CD82 on LT-HSCs decreased, leading to cell-cycle entry, proliferation, and differentiation. A similar interaction appears to be relevant for human HSPCs. Thus, CD82 is a functional surface marker of LT-HSCs that maintains quiescence through interaction with DARC-expressing macrophages in the BM stem cell niche.

Homozygous loss of mouse tetraspanin CD82 enhances integrin αIIbß3 expression and clot retraction in platelets.

Integrin αIIbß3 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 αIIbß3 outside-in signaling. Conversely, mild but enhanced integrin αIIbß3 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 αIIbß3 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 αIIbß3 expression, cell adhesion, and tyrosine signaling.

Loss of tumor suppressors KAI1 and p27 identifies a unique subgroup of primary melanoma patients with poor prognosis.

Primary melanoma, a highly aggressive malignancy, exhibits heterogeneity in biologic behaviors, clinical characteristics, metastasis potential and mortality. The present study sought to identify the molecular signatures that define a subgroup of primary melanomas with high risks of metastasis and mortality. First, we identified the markers that best differentiated metastatic melanomas from primary melanomas by examining the expression of seven previously reported biomarkers (BRAF, Dicer, Fbw7, KAI1, MMP2, p27 and Tip60) in a training cohort consisting of 145 primary melanomas and 105 metastatic melanomas. KAI1 and p27, both tumor suppressors, emerged as best candidates. Loss of both tumor suppressors occurred in the majority (74.29%) of metastatic melanomas. Further, a subset (metastatic like, or "ML", 33.10%) of primary melanomas also lost these two tumor suppressors. Kaplan-Meier analysis indicated that ML subgroup of primary melanoma patients had much worse 5 year survival compared with other primary melanoma patients (P = 0.002). The result was confirmed in an independent validation cohort with 92 primary melanomas (P = 0.030) and in the combined cohort with 237 melanoma patients (P = 3.00E-4). Additionally, compared to KAI1 and p27 as an individual prognostic marker, the combined signature is more closely associated with melanoma patient survival (P = 0.025, 0.264 and 0.009, respectively). In conclusion, loss of both KAI1 and p27 defines a subgroup of primary melanoma patients with poor prognosis. This molecular signature may help in metastatic melanoma diagnosis and may provide information useful in identifying high-risk primary melanoma patients for more intensive clinical surveillance in the future.