Insufficient folding of type IV collagen and formation of abnormal basement membrane-like structure in embryoid bodies derived from Hsp47-null embryonic stem cells

Abstract

Hsp47 is a molecular chaperone that specifically recognizes procollagen in the endoplasmic reticulum. Hsp47-null mouse embryos produce immature type I collagen and form discontinuous basement membranes. We established Hsp47-/- embryonic stem cell lines and examined formation of basement membrane and production of type IV collagen in embryoid bodies, a model for postimplantation egg-cylinder stage embryos. The visceral endodermal cell layers surrounding Hsp47-/- embryoid bodies were often disorganized, a result that suggested abnormal function of the basement membrane under the visceral endoderm. Rate of type IV collagen secretion by Hsp47-/- cells was fourfold lower than that of Hsp47+/+ cells. Furthermore, type IV collagen secreted from Hsp47-/- cells was much more sensitive to protease digestion than was type IV collagen secreted from Hsp47+/+ cells, which suggested insufficient or incorrect triple helix formation in type IV collagen in the absence of Hsp47. These results indicate for the first time that Hsp47 is required for the molecular maturation of type IV collagen and suggest that misfolded type IV collagen causes abnormal morphology of embryoid bodies.

Figure 1.

VE cell layer shows disorganized morphology in EBs of Hsp47^-/- ES cells. The distribution of Hsp47 (A–C), type IV collagen (D–F, J, and K), and laminin (G–I) in EBs differentiated from Hsp47^-/- and Hsp47^+/+ ES cells at day 8 were analyzed by immunostaining. (A, D, G, and J) Hsp47^+/+. (B, E, H, and K) Hsp47^-/- clone 7. (C, F, and I) Hsp47^-/- clone 10. (L) Percentage of EBs that contained normal morphology of VE cell layers adjacent to BM-like structures (see Materials and Methods for details) was determined. Bars, 100 μm (A–I); and 10 μm (J and K). EE, epiblast epithelium; asterisk, cavity.

Figure 2.

Expression of Hsp47 and type IV collagen in differentiating ES cells and EBs. (A) Expression of Hsp47 correlates with that of type IV collagen in monolayer endodermal cells and in EBs differentiated from Hsp47^+/+ ES cells. Total soluble proteins in monolayers of differentiating Hsp47^+/+ ES cells, Hsp47^+/+ EBs, and BALB/3T3 cells was analyzed by Western blotting by using specific antibodies. (B) Type IV collagen in soluble proteins extracted from monolayers of differentiating Hsp47^+/+ and Hsp47^-/- ES cells were analyzed. (C) Northern blot analysis of type IV collagen mRNA in monolayers of differentiating Hsp47^+/+ and Hsp47^-/- ES cells.

Figure 3.

Rate of type IV collagen secretion is significantly slower in Hsp47^-/- cells than in Hsp47^+/+ cells, as determined by pulse-chase experiment. (A–C) Radioactivity of intracellular and secreted type IV collagen of Hsp47^+/+ cells (A), Hsp47^-/- clone 7 cells (B), and Hsp47^-/- clone 10 cells (C). (D) Relative radioactivity of intracellular type IV collagen was plotted in a logarithmic scale (n = 3). (E) Radioactivity of fibronectin secreted into the medium.

Figure 4.

Type IV collagen secreted from Hsp47^-/- cells is susceptible to protease digestion. Differentiating ES cells were cultured in the presence of l-[2,3-[³H]Pro, and aliquots of medium containing equivalent amounts of TCA-insoluble radioactivity were treated with a mixture of trypsin and chymotrypsin at 37°C for indicated periods. The medium before and after protease digestion was analyzed by SDS-PAGE.

Figure 5.

Type IV collagen secreted from Hsp47^-/- cells exhibits higher affinity to fibronectin than that secreted from Hsp47^+/+ cells. Culture medium containing proteins labeled with l-[2,3-[³H]Prowas mixed with fibronectin-Sepharose beads. After washing, proteins bound to the beads were analyzed by SDS-PAGE. (A) Binding of type IV collagen secreted from Hsp47^+/+ or Hsp47^-/- cells to fibronectin. (B) Binding of type IV collagen secreted from Hsp47^+/+ cells after heat treatment. The input applied to SDS-PAGE was 10% of total sample. (C and D) Quantification of type IV collagen binding to fibronectin beads (including means and standard deviations of four experiments). Type IV collagen binding to BSA-Sepharose is shown by solid bars, and type IV collagen binding to fibronectin-Sepharose is shown by open bars. HT, heat-treatment; I, input; B, fraction bound to BSA-Sepharose; F, fraction bound to fibronectin-Sepharose.