Retracted: Photoactivatable Hsp47: A Tool to Regulate Collagen Secretion and Assembly

Abstract

Collagen is the most abundant structural protein in mammals and is crucial for the mechanical integrity of tissues. Hsp47, an endoplasmic reticulum resident collagen‐specific chaperone, is involved in collagen biosynthesis and plays a fundamental role in the folding, stability, and intracellular transport of procollagen triple helices. This work reports on a photoactivatable derivative of Hsp47 that allows regulation of collagen biosynthesis within mammalian cells using light. Photoactivatable Hsp47 contains a non‐natural light‐responsive tyrosine (o‐nitro benzyl tyrosine (ONBY)) at Tyr383 position of the protein sequence. This mutation renders Hsp47 inactive toward collagen binding. The inactive, photoactivatable protein is easily uptaken by cells within a few minutes of incubation, and accumulated at the endoplasmic reticulum via retrograde KDEL receptor‐mediated uptake. Upon light exposure, the photoactivatable Hsp47 turns into functional Hsp47 in situ. The increased intracellular concentration of Hsp47 results in stimulated secretion of collagen. The ability to promote collagen synthesis on demand, with spatiotemporal resolution, and in diseased state cells is demonstrated in vitro. It is envisioned that photoactivatable Hsp47 allows unprecedented fundamental studies of collagen biosynthesis, matrix biology, and inspires new therapeutic concepts in biomedicine and tissue regeneration.

Exogenous supply of Hsp47 triggers fibrillar collagen deposition in skin cell cultures in vitro

“…The uptake of Hsp47 was tested in fibroblast (NHDF from human skin dermis, L929 from mouse adipose tissue and MEF from mouse embryos), epithelial (HaCaT, human epidermal keratinocytes) and endothelial (HDMEC from human dermis) skin cells. A recombinant EGFPtagged Hsp47 (hereafter referred to as H47 [23]) was used for these studies. The different cell types were seeded at same cell density and incubated with H47 at concentrations between 0.1 µM and 1.0 µM.…”

“…We recently demonstrated that uptake of exogenous Hsp47 could specifically enhance collagen deposition in fibroblast cultures [23]. Uniquely, Hsp47 is a collagen-specific chaperone.…”

“…it stabilizes triple helical of procollagen [24][25][26][27][28], it prevents intracellular procollagen degradation [29][30][31], it is involved in quality control of folded procollagen [31,32], it inhibits procollagen aggregate formation in the Endoplasmic Reticulum (ER) [33,34], and it supports procollagen transport to Golgi apparatus [30] by binding to procollagen in the ER (at neutral pH) and dissociating in the cis-Golgi (at low pH). The involvement of endogenous Hsp47 in the biosynthesis of collagen subtypes I to V has been reported [23,29,35,36]. It is however unclear if exogenous administration of Hsp47 to cells affects the deposition of each collagen subtype to a similar level, or if deposition of certain collagen is preferentially supported.…”

“…Hsp47 is retained in the ER via KDEL receptor mediated transport from the Golgi to the ER [26,29,[41][42][43][44][45]. This KDEL-receptor is also found on the cell membrane, [46] and is critical for the transport of exogenous Hsp47 to the ER of cells in vitro by simple incubation with the protein in solution [23]. Expression levels of KDEL receptor at the cell membrane are celldependent [47,48].…”

“…In the current work we follow-up on our previous demonstration of Hsp47-stimulated deposition of collagen in in vitro in fibroblast cultures [23], and we quantify the cell-and typespecific collagen deposition (fibrillar collagens I, III, V, network collagen IV, and fibrilassociated collagen XII) after Hsp47 stimulation in in vitro cultures of fibroblasts, epithelial and endothelial cells from skin tissue. Lastly, the increase in the collagen deposition upon repetitive delivery of recombinant Hsp47 was also studied.…”