Lysophosphatidic Acid Upregulates Laminin-332 Expression during A431 Cell Colony Dispersal

Abstract

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, survival, wound healing, and tumor invasion through LPA receptors. Previously, we reported that LPA induces A431 colony dispersal, accompanied by disruption of cell-cell contacts and cell migration. However, it remains unclear how LPA affects cell migration and gene expression during A431 colony dispersal. In this paper, we performed cDNA microarray analysis to investigate this question by comparing gene expression between untreated and LPA-treated A431 cells. Interestingly, these results revealed that LPA treatment upregulates several TGF-β1 target genes, including laminin-332 (Ln-332) components (α3, β3, and γ2 chains). Western blot analysis also showed that LPA increased phosphorylation of Smad2, an event that is carried out by TGF-β1 interactions. Among the genes upregulated, we further addressed the role of Ln-332. Real-time PCR analysis confirmed the transcriptional upregulation of all α3, β3, and γ2 chains of Ln-332 by LPA, corresponding to the protein level increases revealed by western blot. Further, the addition of anti-Ln-332 antibody prevented LPA-treated A431 colonies from dispersing. Taken together, our results suggest that LPA-induced Ln-332 plays a significant role in migration of individual cells from A431 colonies.

Figure 1

RNA preparation. A431 cells were incubated for 24 h in serum-deprived culture medium. LPA (2 μM) or PBS (control) was added to medium and incubated for 12 h at 37°C in a humidified, 5% CO₂, 95% air atmosphere. Total RNA was isolated from A431 cells treated with PBS or LPA (2 μM) for 12 hrs, using a Qiagen RNeasy kit. The RNA integrity was measured by the RNA integrity number (RIN) software algorithm designed to classify the eukaryotic total RNA, based on numbering from 1 to 10 (1 indicates the most degraded RNA profile, and 10 indicates the most intact RNA). Electrophoresis of RNA in eukaryote total RNA Nano_DE114000902 resulted in an RNA integrity number (RIN) of 10.

Figure 2

cDNA microarray analysis. Microarray analysis was performed on PBS and LPA-treated samples using Affymetrix Human U133 plus 2.0 chips according to manufacturer's instructions, and data were normalized using the Microarray Suite 5.0 algorithm. Among the upregulated genes induced by LPA, all three subchains of the intact Ln-332 molecule: α3 chain (LAMA3), laminin β3 (LAMB3), and laminin γ2 chain (LAMC2) were significantly increased.

Figure 3

LPA induces phosphorylation of Smad-2 during A431 colony dispersal. A431 cells were serum starved for 24 h and incubated with PBS or LPA (1, 2, or 4 μM) for 1 h. (a, b) Cells were lysed and 100 μg of total proteins were separated on 4–12% NuPAGE gels under reducing conditions. 3T3NIH cell lysate after TGF-β1 treatment was used as a positive control for phosphorylation of Smad2. After separation, the same gel was transferred to a PVDF membrane and blocked with 5% skim milk in 1X TBS and 0.1% Tween 20. After blocking, a pAb against phosphorylated Smad2 (p-Smad2; Ser465/467), a mAb against Smad2/3, or GADPH was added at the diluted ratio of 1 : 1000 and incubated at 4°C overnight. Anti-rabbit IgG or mouse IgG HRP-conjugated antibody was used as a secondary antibody at a diluted ratio of 1 : 1000. The bands were visualized with an ECL plus system. Protein expression levels were quantified from the western blots using ImageJ. Results showed that 4 μM LPA treatment for 12 h significantly enhanced pSmad2 protein expression compared to PBS treatment (N = 3; P =  .003).

Figure 4

RT-PCR analysis of mRNA expression of laminin α 3, β 3, and γ 2 genes in LPA-treated A431 cells. To confirm the results of the previously performed microarrays, RT-PCR analysis was performed as described in Section 2. Briefly, A431 cells were treated with PBS or 2 μM LPA for 12 h, total RNA was extracted for each sample, and cDNA was synthesized using reverse transcriptase. Samples were analyzed on a MiQ machine using FastStart SYBR Green Master Mix. The primer sets used are shown in Table 1. Results showed that LPA treatment increased Ln-332 component, α3, β3, and γ2, expression (by ~2–2.5-fold), although results were not determined to be significantly different than PBS control treatments (N = 3; P =  .11,  .08, and .13, resp.).

Figure 5

LPA enhances laminin-332 protein expression in A431 cells. To examine the expression level of Ln-332 chains in LPA-treated A431 cells, cells were serum starved for 24 h and incubated with PBS or 2 μM LPA for 12 h. (a, b) Cells were then lysed and 100 μg of proteins were separated on an 8% gel under reducing conditions. After separation, the same gel was transferred to a PVDF membrane and blocked with 5% skim milk in 1X TBS and 0.1% Tween 20. After blocking, BM165 (mAb against human laminin α3 chain) H-300 (pAb against human laminin β3 chain), a pAb against rat laminin γ2 chain, or a mAb against actin was added at the diluted ratios of 1 : 1000, 1 : 200, 1 : 2000, and 1 : 10000, respectively, and incubated at 4°C overnight. Anti-rabbit IgG or mouse IgG HRP-conjugated antibody was used as a secondary antibody at the diluted ratio of 1 : 1000. The bands were visualized with an ECL plus system (Perkin Elmer, Waltham, MA). Protein expression levels were quantified using ImageJ. Results showed that LPA treatment consistently increased Ln-332 component expression. Both the Ln-332 α3 and β3 chains were determined to be significantly increased (N = 3, P =  .004), whereas the γ2 chain was only somewhat elevated compared to the PBS control treatment.

Figure 6

Anti-Ln-332 antibody prevents A431 colony from dispersing by LPA treatment. Cell colony dispersal assays were performed similarly to our previous studies (serum-starved A431 cells for 24 h, treated with 2 μM LPA), with the addition of an anti-Ln-332 antibody (BM165). (a, b) We found that anti-Ln-332 antibody significantly blocked dispersal of LPA-treated colonies, compared to those colonies treated with LPA alone (N = 3; P <  .05). LPA-treated A431 colony dispersal percentages in the absence (LPA) or presence of BM165 antibody (LPA + anti-Ln-332) are indicated as light-grey triangles or cross, respectively. These results strongly support a general role for Ln-332 in regulating the “LPA effect”. *P <  .05, Two-way ANOVA (Bonferroni post test).

Figure 7

Hypothetical working model of LPA-induced A431 colony dispersal. We hypothesize that LPA induces Ln-332 expression via TGF-β1 pathway. In our working model, LPA binds to LPA receptors on the A431 cell surface and transactivates TGF-β1 type I receptor (TGFβR1), which phosphorylates receptor-regulated Smad (R-Smad), Smad2, and Smad3. These results are supported by other previous findings [36, 40, 41]. Another potential pathway may lead to the direct induction of phosphorylation of Smad2/3.Phosphorylated Smad2/3 forms complex with Smad4 and translocates into the nucleus [41]. Smad4 functions as a positive transcriptional regulator of Ln-332 [42]. Finally, LPA enhances Ln-332 expression to promote A431 colony dispersal.