Advertisement

Rapamycin selectively inhibits expression of an inducible keratin (K6a) in human keratinocytes and improves symptoms in pachyonychia congenita patients

      Abstract

      Background

      The macrolide sirolimus (rapamycin) selectively blocks translation of mRNAs containing a terminal 5′ oligopyrimidine (TOP) tract by altering the activity of mammalian target of rapamycin (mTOR) and inhibiting downstream mTOR pathway components involved in TOP mRNA translation. The skin disorder pachyonychia congenita (PC) is caused by mutations in the inducible keratins (K) including K6a, K6b, K16 and K17. Published sequence data suggest the 5′ untranslated regions of K6a and K6b mRNAs contain 5′ TOP motifs and therefore may be sensitive to rapamycin treatment.

      Objective

      Determine if mTOR inhibitors (rapamycin, temsirolimus or everolimus) are viable drug candidates for treatment of PC and other disorders caused by inappropriate expression of K6a and K6b.

      Methods

      5′ RACE analysis was used to map the transcriptional start sites for K5, K6a, K6b, K14, K16 and K17. The sensitivity of these keratins to mTOR inhibitors was determined by Western and qPCR analysis following treatment of a human HaCaT keratinocyte cell line with rapamycin, temsirolimus or everolimus. A small off-label study was undertaken using orally administered rapamycin in three PC patients and the effects were monitored by clinical examination, photography, a validated Dermatology Life Quality Index (DLQI) and a pain and activity diary.

      Results

      Sequence comparison and 5′ RACE analysis of the 5′ untranslated regions of K6a and K6b revealed putative TOP regulatory elements. Treatment of a human HaCaT keratinocyte cell line with mTOR inhibitors (rapamycin, temsirolimus or everolimus) resulted in selective K6a repression. Furthermore, treatment of this HaCaT cell line with siRNAs targeting components of the mTOR pathway altered the levels of K6a expression. To test the ability of rapamycin to ameliorate PC symptoms, an off-label study was conducted. PC patient clinical responses to oral rapamycin showed a therapeutic response in callus character as well as subjective improvement. Of particular note, rapamycin greatly reduced the presence of painful cutaneous thromboses after reaching therapeutic serum levels. The well-known rapamycin side effects led to the early withdrawal of all of the patients from the study.

      Conclusion

      Rapamycin selectively blocks K6a expression in human keratinocytes. The improvement of symptoms in PC patients following rapamycin treatment suggests rapamycin (or rapamycin analogs) may be a therapeutic option, particularly if topical formulations can be developed that avoid the side effects associated with systemic administration.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Journal of Dermatological Science
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Tyner A.L.
        • Fuchs E.
        Evidence for posttranscriptional regulation of the keratins expressed during hyperproliferation and malignant transformation in human epidermis.
        J Cell Biol. 1986; 103: 1945-1955
        • Takahashi K.
        • Paladini R.D.
        • Coulombe P.A.
        Cloning and characterization of multiple human genes and cDNAs encoding highly related type II keratin 6 isoforms.
        J Biol Chem. 1995; 270: 18581-18592
        • Yamashita R.
        • Suzuki Y.
        • Takeuchi N.
        • Wakaguri H.
        • Ueda T.
        • Sugano S.
        • et al.
        Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics.
        Nucleic Acids Res. 2008; 36: 3707-3715
        • Hay N.
        • Sonenberg N.
        Upstream and downstream of mTOR.
        Genes Dev. 2004; 18: 1926-1945
        • Meyuhas O.
        • Hornstein E.
        Translational control of TOP mRNAs.
        in: Sonenberg N. Hershey J.W.B. Mathews M.B. Translational control of gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 671-693
        • Kaspar R.L.
        • Kakegawa T.
        • Cranston H.
        • Morris D.R.
        • White M.W.
        A regulatory cis element and a specific binding factor involved in the mitogenic control of murine ribosomal protein L32 translation.
        J Biol Chem. 1992; 267: 508-514
        • Avni D.
        • Shama S.
        • Loreni F.
        • Meyuhas O.
        Vertebrate mRNAs with a 5′-terminal pyrimidine tract are candidates for translational repression in quiescent cells: characterization of the translational cis-regulatory element.
        Mol Cell Biol. 1994; 14: 3822-3833
        • Jefferies H.B.
        • Reinhard C.
        • Kozma S.C.
        • Thomas G.
        Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family.
        Proc Natl Acad Sci USA. 1994; 91: 4441-4445
        • Terada N.
        • Patel H.R.
        • Takase K.
        • Kohno K.
        • Nairn A.C.
        • Gelfand E.W.
        Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins.
        Proc Natl Acad Sci USA. 1994; 91: 11477-11481
        • Zhu J.
        • Spencer E.D.
        • Kaspar R.L.
        Differential translation of TOP mRNAs in rapamycin-treated human B lymphocytes.
        Biochim Biophys Acta. 2003; 1628: 50-55
        • Raught B.
        • Gingras A.C.
        • Sonenberg N.
        The target of rapamycin (TOR) proteins.
        Proc Natl Acad Sci USA. 2001; 98: 7037-7044
        • Kawasome H.
        • Papst P.
        • Webb S.
        • Keller G.M.
        • Johnson G.L.
        • Gelfand E.W.
        • et al.
        Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity.
        Proc Natl Acad Sci USA. 1998; 95: 5033-5038
        • Jefferies H.B.
        • Fumagalli S.
        • Dennis P.B.
        • Reinhard C.
        • Pearson R.B.
        • Thomas G.
        Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k.
        EMBO J. 1997; 16: 3693-3704
        • Sonenberg N.
        eIF4E, the mRNA cap-binding protein: from basic discovery to translational research.
        Biochem Cell Biol. 2008; 86: 178-183
        • Smith F.J.D.
        • Kaspar R.L.
        • Schwartz M.E.
        • McLean W.H.I.
        • Leachman S.A.
        Pachyonychia congenita.
        GeneReviews. 2006;
        • Leachman S.A.
        • Kaspar R.L.
        • Fleckman P.
        • Florell S.R.
        • Smith F.J.
        • McLean W.H.
        • et al.
        Clinical and pathological features of pachyonychia congenita.
        J Invest Dermatol Symp Proc. 2005; 10: 3-17
        • McLean W.H.
        • Rugg E.L.
        • Lunny D.P.
        • Morley S.M.
        • Lane E.B.
        • Swensson O.
        • et al.
        Keratin 16 and keratin 17 mutations cause pachyonychia congenita.
        Nat Genet. 1995; 9: 273-278
        • Boukamp P.
        • Petrussevska R.T.
        • Breitkreutz D.
        • Hornung J.
        • Markham A.
        • Fusenig N.E.
        Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.
        J Cell Biol. 1988; 106: 761-771
        • Hickerson R.P.
        • Smith F.J.
        • Reeves R.E.
        • Contag C.H.
        • Leake D.
        • Leachman S.A.
        • et al.
        Single-nucleotide-specific siRNA targeting in a dominant-negative skin model.
        J Invest Dermatol. 2008; 128: 594-605
        • Zhu J.
        • Hayakawa A.
        • Kakegawa T.
        • Kaspar R.L.
        Binding of the La autoantigen to the 5′ untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.
        Biochim Biophys Acta. 2001; 1521: 19-29
        • Randle R.A.
        • Raguz S.
        • Higgins C.F.
        • Yague E.
        Role of the highly structured 5′-end region of MDR1 mRNA in P-glycoprotein expression.
        Biochem J. 2007; 406: 445-455
        • Michlewski G.
        • Sanford J.R.
        • Caceres J.F.
        The splicing factor SF2/ASF regulates translation initiation by enhancing phosphorylation of 4E-BP1.
        Mol Cell. 2008; 30: 179-189
        • Wong P.
        • Coulombe P.A.
        Loss of keratin 6 (K6) proteins reveals a function for intermediate filaments during wound repair.
        J Cell Biol. 2003; 163: 327-337
        • Smith F.J.
        • Hickerson R.P.
        • Sayers J.M.
        • Reeves R.E.
        • Contag C.H.
        • Leake D.
        • et al.
        Development of therapeutic siRNAs for pachyonychia congenita.
        J Invest Dermatol. 2008; 128: 50-58
        • Wong P.
        • Domergue R.
        • Coulombe P.A.
        Overcoming functional redundancy to elicit pachyonychia congenita-like nail lesions in transgenic mice.
        Mol Cell Biol. 2005; 25: 197-205
      1. PDF. Physicians’ Desk Reference. 61 ed.; 2007.

        • Lewis V.
        • Finlay A.Y.
        10 years experience of the Dermatology Life Quality Index (DLQI).
        J Invest Dermatol Symp Proc. 2004; 9: 169-180
        • Finlay A.Y.
        • Khan G.K.
        Dermatology Life Quality Index (DLQI)—a simple practical measure for routine clinical use.
        Clin Exp Dermatol. 1994; 19: 210-216
        • Mahe E.
        • Morelon E.
        • Lechaton S.
        • Kreis H.
        • De Prost Y.
        • Bodemer C.
        Sirolimus-induced onychopathy in renal transplant recipients.
        Ann Dermatol Venereol. 2006; 133: 531-535
        • Leachman S.A.
        • Hickerson R.P.
        • Hull P.R.
        • Smith F.J.
        • Milstone L.M.
        • Lane E.B.
        • et al.
        Therapeutic siRNAs for dominant genetic skin disorders including pachyonychia congenita.
        J Dermatol Sci. 2008; 51: 151-157
        • Jimenez-Diaz L.
        • Geranton S.M.
        • Passmore G.M.
        • Leith J.L.
        • Fisher A.S.
        • Berliocchi L.
        • et al.
        Local translation in primary afferent fibers regulates nociception.
        PLoS ONE. 2008; 3: e1961
        • Guba M.
        • von Breitenbuch P.
        • Steinbauer M.
        • Koehl G.
        • Flegel S.
        • Hornung M.
        • et al.
        Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor.
        Nat Med. 2002; 8: 128-135
        • Phung T.L.
        • Ziv K.
        • Dabydeen D.
        • Eyiah-Mensah G.
        • Riveros M.
        • Perruzzi C.
        • et al.
        Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.
        Cancer Cell. 2006; 10: 159-170
        • Meric-Bernstam F.
        • Gonzalez-Angulo A.M.
        Targeting the mTOR signaling network for cancer therapy.
        J Clin Oncol. 2009; 27: 2278-2287
        • Ormerod A.D.
        • Shah S.A.
        • Copeland P.
        • Omar G.
        • Winfield A.
        Treatment of psoriasis with topical sirolimus: preclinical development and a randomized, double-blind trial.
        Br J Dermatol. 2005; 152: 758-764
        • Simpson D.
        • Curran M.P.
        Temsirolimus: in advanced renal cell carcinoma.
        Drugs. 2008; 68: 631-638
        • Dunn C.
        • Croom K.F.
        Everolimus: a review of its use in renal and cardiac transplantation.
        Drugs. 2006; 66: 547-570
        • Lorber M.I.
        • Mulgaonkar S.
        • Butt K.M.
        • Elkhammas E.
        • Mendez R.
        • Rajagopalan P.R.
        • et al.
        Everolimus versus mycophenolate mofetil in the prevention of rejection in de novo renal transplant recipients: a 3-year randomized, multicenter, phase III study.
        Transplantation. 2005; 80: 244-252
        • Bottiger Y.
        • Sawe J.
        • Brattstrom C.
        • Tollemar J.
        • Burke J.T.
        • Hass G.
        • et al.
        Pharmacokinetic interaction between single oral doses of diltiazem and sirolimus in healthy volunteers.
        Clin Pharmacol Ther. 2001; 69: 32-40