Long non-coding RNA MT1DP shunts the cellular defense to cytotoxicity through crosstalk with MT1H and RhoC in cadmium stress

Metallothioneins (MTs) are known to protect cells against oxidative stress, especially providing protection against cadmium (Cd) toxicity in hepatocytes. There are various gene variants and

pseudogenes for MTs; however, there is little understanding on the functions of those non-coding MT members that are known to be expressed as long non-coding RNAs (lncRNAs) nowadays.

Different from most protein-coding MT members, MT1DP was here found that remarkably induced to provoke cytotoxicity in hepatocytes in response to Cd treatment. MT1DP exerted such a

pro-apoptotic function in Cd-treated hepatocytes through interacting with two partners: RhoC and MT1H. On one hand, MT1DP interacted with RhoC protein to increase the latter’s stability by

preventing lysosome-dependent protein degradation. Therefore, upon Cd stress, MT1DP/RhoC complex was quickly reinforced to activate RhoC-CCN1/2-AKT signaling and potentiate Ca2+ influx,

leading to enhanced Cd uptake and elevated Cd toxicity. On the other hand, MT1H, a protein-coding member of the MT family with little known function, was found to quickly respond to Cd

exposure along with MT1DP. Mechanistically, MT1H and MT1DP were uncovered to mutually protect each other through a reciprocal ceRNA mechanism, building up a positive feedback loop to enforce

MT1DP-conducted signaling upon Cd exposure. Moreover, MT1DP was found to contribute much more to the activation of RhoC-CCN1/2-AKT signaling than MT1H. Considered together, we here unveiled

a mystery whether a pseudogene within the MT family, MT1DP, has actual biological functions in regulating Cd-induced cellular defense. Our findings unearthed an important role of pseudogene

MT1DP in calibrating the cellular machinery to switch the cellular defense to cytotoxicity through crosslinking an interplay between its two partners, namely MT1H and RhoC, under cadmium

stress.

Mammals have developed evolutionarily conserved intricate defense mechanisms against stress in response to toxic substances, such as antioxidant agents, detoxification enzymes, pro-survival

signaling, autophagy and metal-binding proteins1,2,3,4. Thus far, metallothioneins (MTs) have been extensively investigated for their protection from cadmium (Cd) toxicity5, 6. The pivotal

role of MTs in Cd detoxification has been established prominently due to Cd sequestration through their high-affinity binding inside cells, resulting in reduced Cd mass to prevent damage to

cellular organelles5, 6. This mechanism is further verified by MT-transgenic mice, as increased MT expression endowed mice with enhanced tolerance to Cd toxicity and MT-null mice were

contrastively more vulnerable7, 8. There are at least 16 members in the MT family including 12 protein-coding variants (i.e., MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1L, MT1M, MT1X, MT2, MT3

and MT4) and 4 pseudogenes (namely MT1CP, MT1DP, MT1IP, and MT1JP) without protein-coding functionality in the human genome. Yet, the inter-regulation among these members (e.g., synergism

and antagonism) and particularly their biological functions of non-coding pseudogenes (now known as long non-coding RNAs, lncRNAs) are still elusive.

As a subtype of non-coding RNAs, lncRNAs are transcribed in sense or antisense to protein-coding genes, with the length >200 nucleotides9, 10. Accumulating studies have documented the

necessary contribution of lncRNAs to fundamental physiological homeostasis and versatile biological functions, whereas deregulated expression for certain lncRNAs may cause diverse

pathologies including cancers and diabetes11,12,13. LncRNAs implement their functions through modulation of gene expression (such as chromatin remodeling and posttranscriptional modification

of target mRNAs) and regulation of protein activity via physical interaction14, 15. Historically, pseudogenes were defined as “junk” DNA, such as pseudogenes in the MT family, due to their

lack of protein-coding functionality, whereas recent reports suggested that many pseudogenes express non-coding RNAs, including lncRNAs16. However, the biological significance of most

non-coding RNAs transcribed from pseudogenes is largely unknown16, 17.

Given that the pseudogenes in the MT family themselves are lncRNAs, we postulated that these lncRNAs may antagonize or synergize the real MTs at the transcriptional or posttranscriptional

level in order to orchestrate cellular defense and cytotoxicity. In the current study, one of the pseudogenes, MT1DP, was found to be dramatically induced in hepatocytes by Cd treatment.

Different from MT1/2, MT1DP is present in human genome but not in mouse genome18. It was reported that MT1DP acted as a tumor suppressor through negatively regulating YAP and Runx2 to

promote apoptosis of liver cancer cells19; however, the biological functions of MT1DP are still almost unexploited thus far, with no clue linking to Cd toxicity. Our results unearthed a

vital role of lncRNA MT1DP in antagonizing the cytoprotective role of most MT members to enhance Cd-induced toxicity in hepatocytes through physically interacting with RhoC (Ras homolog gene

family, member C). Stabilized MT1DP/RhoC complex thereafter activated CCN1/2-AKT signaling to promote cell death dependent on elevated Cd uptake. Meanwhile, the function of MT1DP was

further enhanced by a MT family member, MT1H, which functioned as a competing endogenous RNA (ceRNA) to block miR-214-conducted suppression on MT1DP. These findings together unveiled the

molecular bases underlying MT-associated cytotoxicity versus cellular defense under Cd stress. This study would open a path to understand the regulation of a pseudogene in a form of lncRNA

on its according protein-coding gene.

To shed light on the interplay among the MT family members, we first compared the expression profiles of 10 real MT1/2 genes and 4 pseudogenes in hepatocytes upon Cd treatment. As shown in

Fig. 1a, the 10 protein-coding members were markedly stimulated in HepG2 cells by Cd at 20 μM, analogous to previous studies20, 21. The induction of protein-coding members was confirmed at

the protein levels, as evidenced by the western blot results (Fig. 1a), supporting of a critical role of MT1/2 against Cd toxicity6. Moreover, all non-coding members, MT1CP, MT1DP, MT1IP,

and MT1JP were also induced by Cd, especially for MT1DP with >100-fold increase (Fig. 1a, P