In silico analysis of HACS1 expression from microarray hybridization data of lymphocyte samples. (A) HACS1 clustered together with known signaling proteins and uncharacterized genes and ESTs. (B) HACS1 can be up-regulated in B cells under different culture conditions such as addition of IL-4, anti-IgM, and CD40L. Data was mined from Alizadeh et al. (4) through SOURCE (5). (C) Gene-specific RT-PCR analysis of HACS1 and related gene HACS2/Sly shows only HACS1 can be up-regulated by IL-4, confirming the microarray data.

Hacs1 was up-regulated by IL-4 and other B cell activators in B220⁺ murine splenocytes. (A) Purified murine B220⁺ splenocytes were incubated with an increasing amount of IL-4 overnight or (B) incubated with 10 ng/ml of IL-4 for the indicated time, and then cells were harvested, lysed, and analyzed by Western blot. (C) Immunofluorescence staining of Hacs1 protein in B220⁺ mouse spleen B cells in vitro stimulated with IL-4. The up-regulation of Hacs1 expression (green) was evident by 6 h poststimulation as shown on the left (green). The nuclei were stained red with propidium iodide (PPI) as shown on the middle panel. The right panel shows the two stained images merged together. (D) B220⁺ cells were incubated with various B cell activators alone or with IL-4 plus different stimuli for the indicated time, and then expression of Hacs1 was analyzed by Western blot.

IL-4–activated PKCζ and up-regulation of Hacs1 was blocked by inhibition of NF-κB. (A) IL-4 induced phosphorylation of PKCζ in murine splenic B cells. B220⁺ cells were incubated with 10 ng/ml IL-4 for the indicated time, and expression of phosphorylated PKCζ was analyzed by Western blot. (B and C) Inhibition of NF-κB blocked nuclear expression of NF-κB and the subsequent up-regulation of Hacs1 by IL-4. B220⁺ cells were pretreated with or without Bay11-7082 or PDTC for 15 min and then incubated with 10 ng/ml IL-4 for the indicated time. The nuclear extracts or whole cell lysates were prepared and analyzed for expression of NF-κB p50 and Stat6 in nuclei (B) or for expression of Hacs1 in whole cells (C) by Western blot.

The role of HACS1 is likely involved in B cell activation and differentiation. (A) Effects of HACS1 on IL-4 and CD-40–driven B220⁺ cell proliferation. Murine splenic B220⁺ cells were infected as described in Materials and Methods. GFP⁺ cells were sorted, and 3 × 10⁴ cells were seeded in triplicate in 96-well plates with or without IL-4 (10 ng/ml) or anti-CD40 (5 μg/ml) for 48 h, followed by an MTT assay. The data is representative of three independent experiments. (B and C) Effects of HACS1 on the surface phenotype of B220⁺ cells. The cells treated as above in triplicates were harvested and combined and stained with PE-conjugated antibodies against CD23 and CD138. Flow cytometry data is converted to mean fluorescence intensity of the surface expression of living cells. The data is representative of three independent experiments. (D) HACS1 enhanced IgM secretion in vitro. Cell culture supernatant was collected from triplicates of each group above, and IgM levels were measured by ELISA assay. The mean of triplicates is shown, which is representative of two independent experiments. (E) Expression of HACS1 in transduced B220⁺ cells (48 h postinfection) was confirmed by Western blot analysis. (F) Expression of Xbp-1 was enhanced in HACS1-transduced B220⁺ cells. Briefly, HACS1 and control virus-infected B220⁺ cells were harvested after 48 h of infection, and total RNA was extracted, quantitated, and analyzed by RT-PCR. (G) HACS1-specific siRNA knocked down endogenous HACS1 in BJAB cells. BJAB cells were transfected with either control scrambled oligos (C) or HACS1-specific siRNA oligos (H) by electroporation. After 48 h of transfection, cells were treated with indicated reagents for 5 min, and the expression of HACS1 was analyzed by Western blot. (H) Knock down of HACS1 in BJAB cells did not significantly affect cell proliferation stimulated by IL-4. 48 h after transduction of control (C) and HACS1 (H) siRNA oligos, 1 × 10⁴ cells were seeded in triplicate in 96-well plates with or without IL-4 (25 ng/ml) or anti-IgM (5 μg/ml) for 72 h, followed by an MTT assay. The data is representative of two independent experiments.

Immunohistochemistry of mouse spleen and lymph node. Hacs1 is expressed in mouse splenic red pulp and lymph node sinusoids. Low power views of spleen (A) and lymph node (B) stained with anti-HACS1. The white pulp (W) and red pulp (R) in spleen are shown in both magnifications. High power views of spleen and lymph node stained with anti-HACS1 are shown in the bottom panel. Hacs1 is expressed in the cytoplasm of most of the positive cells but is also present in nuclei of some cells (inset in spleen bottom panel).

HACS1 was up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral B cells. (A) Purified human peripheral CD19⁺ cells were incubated with 10 ng/ml IL-4 or 1 μg/ml anti-IgM or 1 μg/ml anti-CD40 overnight, and then cells were harvested and lysed and HACS1 expression was analyzed by Western blot. (B) Western blot analysis of HACS1 expression in CD19⁺ cells induced by IL-13 (10 ng/ml overnight) and in human HL cell lines L1236, HDLM2, and L428 and L540. K562 (erythroleukemia) served as a positive control (1).

Up-regulation of Hacs1 by IL-4 via Stat6, PI 3-kinase, and PKC pathways. (A) B220⁺ cells were incubated with 10 ng/ml IL-4 for the indicated time, and the tyrosine phosphorylation of Stat6 and Akt were analyzed by Western blot. (B) Stat6 is necessary for up-regulation of Hacs1 by IL-4. B220⁺ cells were purified from Stat6-deficient mice (Stat6^−/−) and control mice (Balb/c and J129) and then incubated with IL-4 (10 ng/ml) for 18 h. Expression of Hacs1 was analyzed by Western blot. (C) Inhibition of PI 3-kinase and PKC impaired up-regulation of Hacs1 by IL-4 in B220⁺ murine splenocytes. B220⁺ cells were pretreated without or with different inhibitors (100 nM wortmannin [wort], 10 μM PD98059 [PD], 20 nM rapamycin [Rapa], and 5 μm Bis I) for 15 min, and then 10 ng/ml IL-4 were added and incubated for 8 h. The cells were lysed and analyzed by Western blot.

HACS1 associates with phosphotyrosine-containing proteins in stimulated B cells. (A) Lysates from human BJAB cells were immunoprecipitated with anti-HACS1 antibody and preimmune serum control after stimulation with or without goat anti–human IgM for 5 min. The presence of tyrosine-phosphorylated proteins associated with HACS1 were assessed by immunoblotting with an antiphosphotyrosine antibody (4G10). Reblotting shows the level of HACS1 in the BJAB cell line. (B) Human BJAB cells were electroporated with the cytoplasmic tail of PIR-B using a pEF(HA)₂PIR-B construct or the pEF(HA)₂ vector alone. After stimulation with or without goat anti–human IgM for 5 min, immunoprecipitation was performed with anti-HACS1 and control IgG antibodies. Western blotting was performed with anti-HA antibody (top) and anti-HACS1 antibody (bottom), showing that the cytoplasmic tail of PIR-B binds to HACS1 in vitro.