PDF - 96K, Legend for Supplemental Figures S1-S9.

PDF - 82K, Description of additional methods and procedures used in the study.

PDF - 1790K, ALCAM expression is upregulated in response to the loss of ECadherin (S1); TGFbeta-induced shedding (S2); ALCAM serum half-life in mice (S3); ALCAM shedding by prostate and breast cancer cells in vitro and in vivo (S4); Compound-32/ADAM17 inhibitor dosing in vivo (S5); TGFbeta-induced migration is abrogated by the loss of ALCAM in breast and lung cancer cells (S6); Representative TRAcP stained bone lesion created by PC3 shControl and shALCAM after intratibial injection into immunodeficient mice (S7); Immune response modulation and wound healing does not effect serum ALCAM (S8); Graph represents tumor cell proliferation over time as assessed by total cell counts over 72 hours (S9).

AIMSAccumulating evidence suggest that sarcomere signalling complexes play a pivotal role in cardiomyocyte hypertrophy by communicating stress signals to the nucleus to induce gene expression. Ankyrin repeat domain 1 (ANKRD1) is a transcriptional regulatory protein that also associates with sarcomeric titin; however, the exact role of ANKRD1 in the heart remains to be elucidated. We therefore aimed to examine the role of ANKRD1 in cardiomyocyte hypertrophic signalling.METHODS AND RESULTSIn neonatal rat ventricular myocytes, we found that ANKRD1 is part of a sarcomeric signalling complex that includes ERK1/2 and cardiac transcription factor GATA4. Treatment with hypertrophic agonist phenylephrine (PE) resulted in phosphorylation of ERK1/2 and GATA4 followed by nuclear translocation of the ANKRD1/ERK/GATA4 complex. Knockdown of Ankrd1 attenuated PE-induced phosphorylation of ERK1/2 and GATA4, inhibited nuclear translocation of the ANKRD1 complex, and prevented cardiomyocyte growth. Mice lacking Ankrd1 are viable with normal cardiac function. Chronic PE infusion in wild-type mice induced significant cardiac hypertrophy with reactivation of the cardiac fetal gene program which was completely abrogated in Ankrd1 null mice. In contrast, ANKRD1 does not play a role in haemodynamic overload as Ankrd1 null mice subjected to transverse aortic constriction developed cardiac hypertrophy comparable to wild-type mice.CONCLUSIONOur study reveals a novel role for ANKRD1 as a selective regulator of PE-induced signalling whereby ANKRD1 recruits and localizes GATA4 and ERK1/2 in a sarcomeric macro-molecular complex to enhance GATA4 phosphorylation with subsequent nuclear translocation of the ANKRD1 complex to induce hypertrophic gene expression.

Abstract The dissemination of prostate cancer to bone is a common, incurable aspect of advanced disease. Prevention and treatment of this terminal phase of prostate cancer requires improved molecular understanding of the process as well as markers indicative of molecular progression. Through biochemical analyses and loss-of-function in vivo studies, we demonstrate that the cell adhesion molecule, activated leukocyte cell adhesion molecule (ALCAM), is actively shed from metastatic prostate cancer cells by the sheddase ADAM17 in response to TGF-β. Not only is this posttranslational modification of ALCAM a marker of prostate cancer progression, the molecule is also required for effective metastasis to bone. Biochemical analysis of prostate cancer cell lines reveals that ALCAM expression and shedding is elevated in response to TGF-β signaling. Both in vitro and in vivo shedding is mediated by ADAM17. Longitudinal analysis of circulating ALCAM in tumor-bearing mice revealed that shedding of tumor, but not host-derived ALCAM is elevated during growth of the cancer. Gene-specific knockdown of ALCAM in bone-metastatic PC3 cells greatly diminished both skeletal dissemination and tumor growth in bone. The reduced growth of ALCAM knockdown cells corresponded to an increase in apoptosis (caspase-3) and decreased proliferation (Ki67). Together, these data demonstrate that the ALCAM is both a functional regulator as well as marker of prostate cancer progression. Cancer Res; 74(5); 1404–15. ©2014 AACR.

The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs.

Hypertrophic cardiomyocyte growth occurs in response to various stress stimuli including biomechanical stress and neurohormonal factors. Accumulating evidence suggest that sarcomere signaling complexes play a pivotal role in the cardiomyocyte hypertrophic response by transmitting signals to the nucleus to induce gene expression. Cardiac ankyrin repeat protein (CARP, Ankrd1) is a transcriptional regulatory protein that also associates with the titin I-band spring domain, however the exact role of CARP in the heart remains to be elucidated. We report that CARP directly interacts with mitogen activated protein kinase ERK1/2 and cardiac transcription factor GATA4. Phenylephrine (PE) stimulation in cardiomyocytes induced ERK1/2 and GATA4 to transiently co-localize with sarcomeric CARP, followed by translocation of CARP and GATA4 to the nucleus. Four-and-a-half-LIM (FHL) domains proteins are part of a sarcomeric ERK2 sensory complex and knockdown of CARP by small interfering RNA (siRNA) resulted in disruption of FHL1 and FHL2. Moreover, loss of CARP attenuated PE-induced phosphorylation of ERK1/2 and GATA4, decreased GATA4 DNA binding, and prevented PE-induced cardiomyocyte growth. Mice lacking CARP have decreased FHL1 levels, and PE stimulation in wild-type mice resulted in elevated GATA4 phosphorylation and a hypertrophic response, which were completely abrogated in CARP-KO mice. We demonstrate that CARP plays an important role in PE-induced hypertrophic signaling by recruiting ERK2 and GATA4 into a titin I-band macro-molecular complex to induce GATA4 activation, followed by translocation of CARP and GATA4 to the nucleus to enhance GATA4 DNA binding and hypertrophic gene expression. Loss of CARP destabilizes FHL1 and FHL2, resulting in disruption of the PE-induced sarcomeric complex and abrogation of the cardiomyocyte hypertrophic response. These data reveal a novel role for sarcomeric titin I-band as a transcription factor activation hub that induces downstream nuclear signaling in response to agonist-induced hypertrophic stimuli.

The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1(fl/fl) mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1(-/-) mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenovirat expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1(-/-) mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1(-/-) mice. More important, Ankrd1(-/-) fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenovirat infection stimulated both collagen gel contraction and actin fiber organization. These in vitro data were consistent with in vivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both in vitro and in vivo.

Doxorubicin (adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin, however, the mechanism(s) of doxorubicin-induced CARP suppression and its specific role in cardiomyocyte biology remains to be elucidated. In this study, we report that treatment of cardiomyocytes with doxorubicin resulted in complete suppression of CARP promoter activity, decreased CARP protein levels, and marked sarcomere disarray. Transfection of CARP siRNA in cardiomyocytes resulted in a complete depletion of CARP and significant disruption of sarcomere ultrastructure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. GATA4 has previously been shown to regulate CARP, thus we examined the role of GATA4 in doxorubicin-induced CARP depletion. Cardiomyocytes treated with doxorubicin show a concomitant depletion of CARP and GATA4 protein levels. GATA4 siRNA inhibits while GATA4 overexpression enhances CARP promoter activity in cardiomyocytes. Both GATA4 and CARP siRNA significantly repressed titin and actin promoter activity. These data show that in cardiomyocytes transcription factor GATA4 is upstream of CARP and that doxorubicin induces a rapid down-regulation of GATA4 resulting in inhibition of CARP transcription. Our data further support a role for a GATA4/CARP signaling axis in sarcomere maintenance and that suppression of this pathway contributes, in part, to the overall pathophysiology of doxorubicin cardiomyopathy.

Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

Gene expression profiling of mouse skin wounds has led to the discovery of numerous target genes that may have therapeutic or diagnostic value. Cardiac ankyrin repeat protein (CARP, ankrd1) expression is markedly and persistently elevated in cutaneous wounds. This article provides a summary and perspective on CARP with particular reference to tissue repair. CARP plays a role as a nuclear transcription co-factor in many cell types including vascular endothelium. It is also a structural component of the sarcomere. CARP transcription is active in cardiogenesis and muscle injury, and in the latter context, ankrd1 is under complex regulation by cytokines, hypoxia, doxorubicin and other forms of stress. We have investigated the pharmacology of CARP in wounds by adenoviral gene transfer. Overexpression of CARP leads to a high vascular density, consistent with multiple effects on endothelial behavior. The activity of CARP makes it a novel candidate gene in tissue repair.

Gene expression profiling of mouse skin wounds has led to the discovery of numerous target genes that may have therapeutic or diagnostic value. Among these, cardiac ankyrin repeat protein (CARP, ankrd1) expression was markedly and persistently elevated in several cutaneous compartments. This review summarizes the current state of knowledge of CARP and its regulation in biological systems. In addition to its role as a nuclear transcription cofactor in many cell types including vascular endothelium, CARP is also a structural component of the sarcomere. CARP transcripts are prominent in cardiogenesis and muscle injury, and they are under complex regulation by cytokines, hypoxia, doxorubicin, and other forms of stress. CARP overexpression in wounds by adenoviral gene transfer leads to a high vascular density, and CARP exerts effects on endothelial behavior. The unusual cellular distribution and actions of CARP make it a novel candidate gene in tissue repair.