Chino Kumagai

Differentiation-dependent expression of laminin-8 (α4β1γ1) mRNAs in mouse 3T3-L1 adipocytes☆

Abstract We report that laminin-8 (α4β1γ1) is the specific isoform of laminin synthesized in adipocytes. Reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from mouse 3T3-L1 cells with paired primers for α1, α2, α3, α4, α5, β1, β2, β3, γ1 and γ2 laminins yielded amplified fragments only for α4, β1 and γ1. A polyclonal antibody against mouse laminin-1 (α1β1γ1) precipitated α4 in addition to β1 and γ1, while the antibody against a deduced peptide sequence of mouse α4 precipitated β1 and γ1 in addition to α4. Thus, laminin-8 (α4β1γ1) is the only isoform expressed in 3T3-L1 cells. Northern blots showed that the levels of α4, β1 and γ1 mRNAs increased 2.5-fold during adipose conversion of 3T3-L1 cells. A 1062 bp cDNA fragment cloned by RT-PCR demonstrated a polymorphism in the mouse α4 gene which would lead to five amino acid changes in the domain G.

Three heterotrimeric laminins produced by human keratinocytes

Laminins are a family of glycoproteins composed of α,β and γ chains. Five α(α1-α5), three β (β1–β3) and twoγ (γ1 and γ2) chains have been cloned fromhuman and their replaceable assembly into heterotrimers producesthe variety of laminins. Reverse transcription-polymerase chainreaction of mRNAs showed that human keratinocytes express theα3, α5, β1, β3, γ1 andγ2 genes at high level among the ten cloned lamininchains. Western blot and immunoprecipitation of the cell lysatewith antiserum directed against mouse laminin-1(α1β1γ1) detected two trimers with thecomposition of αxβ1γ1 (probablylaminin-10 with the composition of α5β1γ1and αyβ1γ1. Meanwhile, antiserum directedagainst a synthetic peptide of human α3 detected onlyα3β3γ2 trimer (laminin-5). We thus show thatkeratinocytes produce three heterotrimeric laminins. We couldnot detect the assembly of α3 with β1 and γ1chains to form α3β1γ1 (laminin-6) in keratinocytes.

Potential molecular chaperones involved in laminin chain assembly.

To explore potential molecular chaperones involved in the intracellular assembly of laminin chains, bovine aortic endothelial cells were treated with a thiol cleavable divalent cross-linking reagent, dithio-bis-(succinimidylpropionate), and cellular proteins cross-linked to laminin chains were co-immunoprecipitated with anti-laminin antiserum. Sodium dodecylsulfate (SDS) gel electrophoresis of the precipitate under reducing condition showed polypeptides with estimated sizes of 80, 60 and 50 kDa together with laminin chains. Two dimensional electrophoresis, in which non-reducing and reducing SDS electrophoresis were combined, suggested that many molecules of these polypeptides were cross-linked to each laminin chain. Sepharose CL-4B beads conjugated with E8 fragment of mouse laminin-1 was prepared. Affinity chromatography with the beads of microsomal proteins from rat liver showed that Bip and HSP70 associated to laminin chains and dissociated upon ATP hydrolysis. Protein-disulfide isomerase also showed affinity to the column. GRP94 and calnexin showed strong affinity and were washed out only with a detergent solution. Thus, many molecular chaperones are suggested to be involved in the intracellular assembly of laminin chains.

LAMININ CHAINS EXPRESSED IN HUMAN KERATINOCYTES, MOUSE 3T3-LI ADIPOCYTES AND MOUSE EMBRYONAL CARCINOMA F9

Laminin chain genes expressed in three cultured cell lines were explored by reverse transcription-polymerase chain reaction (RT-PCR) using paired primers designed based on reported cDNA sequences. RT-PCR on mRNA extracted from human keratinocytes gave amplified fragments for γ2, α3, γ1, β1 and γ3 mRNA from mouse 3T3-L1 adipocytes gave the fragments for α4, β and α1 suggesting the assembly of only laminin-8 (α4β1γ1) and the amount of these mRNAs increased depending on the adipocyte differentiation. mRNA from mouse embryonal carcinoma F9 cells gave the fragments for α1, α4, α5, β1 and γ1, and the result remained unchanged by the differentiation of F9 cells into primitive, parietal or visceral endoderm-like cells.

DROSOPHILA CELL LINES AS THE SOURCE OF BASEMENT MEMBRANE COMPONENTS

Drosophila cell lines Kc 167, Schneider (both originated from hemocyte precursor) and Clone 8 (originated from wing disc) secrete large amount of laminin and type IV collagen. In addition to these mammalian equivalents of basement membrane components, they secrete Drosophila specific extracellular matrix proteins such as peroxidasin, glutactin, tiggrin and papilin and unknown polypeptide of 300 kDa. Kc 167 was the best in terms of the productivity and active growth in a serum-free medium (HyQ CCM3). Kc 167 secreted large amount of basement membrane components (1.2 mg from 1 g cells) comparable to that of mouse EHS tumor (6 mg from 1 g tumor). Secreted proteins could be effectively separated by sucrose density sedimentation. Drosophila basement membrane components showed an inhibitory effect to the attachment of bovine aortic endothelial cells to culture dishes.

Specific and interchangeable association of .ALPHA.-helices:Mainly for the formation of laminin isoforms.

Specific and interchangeable association of α-helix chains by hydrophobic and ionic interaction is discussed mainly for the assembly of αn, βn and γn subchains into various laminin isoforms. Sequence of laminin subchains suggests that the hydrophobic surface of α-helix formed by non-polar amino acids at positions a and d in (abcdefg)n heptad repeat is major force of interchain interaction but the ionic edges formed by charged amino acids at positions e and g determine the specificity of interaction.

Subunit Assembly of Laminin Variants in Cultured Animal Cells

Bovine aortic endothelial cells (BAEC) produce two variant forms of laminin with a subunit composition of AB1B2 and A’B1B2. Analyses of the intracellular assembly of these subunits revealed that the B1B2 dimer formed first, and that A or A’ joined to form the AB1B2 or A’B1B2 trimer. Angiostatic steroids shifted the relative size of the A and A’ monomer pool in BAEC, and competition between the A and A’ subunits in joining the B1B2 dimer produced AB1B2 and A’B1B2 in different ratios. This result suggests that subunit replacement is the general mechanism for producing laminin variants by various cells for tissue morphogenesis. When laminin subunits in BAEC were cross-linked with dithio-bis-(succinimidylpropionate)(DSP) and immunoprecipitated with anti-laminin antiserum, monomeric A, A’, B1 and B2 monomers and the B1B2 dimer migrated as extremely large molecules in sodium dodecyl sulfate gel electrophoresis under non-reducing conditions. When the crosslinking disulfide bonds were cleaved under reducing conditions, they migrated as the usual subunits. This result suggests that molecular chaperones were involved in the process of the assembly and replacement of laminin subunits.